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Changes in antigenicity of basement membrane during wound healing
DEVELOPMENTAL
BIOLOGY
Changes
23, 534-5&l
(1970)
in Antigenicity of Basement during Wound Healing
Membrane
LEWIS D. JOHNSON AND G. BARRY PIERCE Department
of Pathology,
Denver, Accepted
University of Colorado Colorado 80220 September
Medical
Center,
10, 1970
INTRODUCTION
Through the isolation and manipulation of epithelial tumors in vitro, it has been possible to demonstrate by direct means that epithelial cells synthesize the basement membranes upon which they rest. These have been named epithelial basement membranes’ to distinguish them from those of endothelium and muscle cells, for example. Chemical analyses of epithelial basement membrane produced in vitro or in the ascites (Mukerjee et al., 1965), situations lacking contamination by connective tissues, have confirmed the idea that there was a tropocollagen core in the molecule. To this collagen is linked a mucoprotein (Kefalides, 1968; Spiro, 1967; Spiro and Fukushi, 1969) which is antigenic and serves as a specific marker for all epithelial basement membranes of a particular species (Midgley and Pierce, 1963; Pierce et al., 1964). Physical, chemical, or microbial injury of epithelial cells results in increased synthesis of epithelial basement membrane, an observation compatible with the notion that basement membrane may subserve a protective role for the adjacent cells (Pierce and Nakane, 1969). Little is known about the turnover of basement membrane, although Grill0 and Gross (1967) have demonstrated an enzyme synthesized by epithelial cells at the margins of cutaneous wounds which cleaves the collagen molecule at a specific place, and which has been named “collagenase”. In view of its collagenous component it is conceivable that one of the substrates of this enzyme may be basement membrane. If this were the case, one might expect enzymatic degradation of epithelial basement membrane during the healing of cutaneous wounds. 1 Epithelial basement membrane called basal lamina or adepidermal basement membrane of amphibians, layers of collagen.
applies to what, in the mammal has also been membrane, and it is not to be confused with the which in addition, includes several well ordered 534
BASEMENT
MEMBRANE
IN
WOUND
HEALING
535
A great deal has been learned about wound healing including studies of the mitotic activity and migration of epithelial cells, and the synthesis of collagen by fibroblasts to restore the epithelium and dermis, respectively (reviews by Schilling, 1968; Schmidt, 1968). Despite the extent of the studies, only brief reference has ever been made to epithelial basement membrane during wound healing (Odland and Ross, 1968; Giacometti and Parakkal, 1969). In a study to be reported here, we have examined wounds timewise, paying particular attention to the antigenicity and ultrastructure of the epithelial basement membrane. Epithelial basement membrane antigen disappeared from the wound margin 2 hours after wounding, then epithelium migrated across a substratum of fibrin, serum proteins, and collagen to “close” the wound, and the epithelial basement membrane antigen did not reappear until after the completion of remodeling of the wound epithelium. MATERIALS
AND
METHODS
Male ICR mice, 6-8 weeks old, were prepared by shaving the abdominal wall, and longitudinal incisions were made through all skin layers down to the fascia. Most animals were incised in three places, to ensure that adequate material could be taken from each mouse for both immunochemistry and electron microscopy. Each incision was approximately 2 cm in length and gaped 2 to 3 mm at the widest point. Bleeding was minimal and although sterile procedure was not followed, infected wounds were infrequent and were not examined. The wounds were allowed to heal without closure or dressings. Mice were sacrificed at 0, 1, 2, 4, 8, 16, and 24 hours, then at 24-hour intervals through the tenth day. To prevent curling of the specimen and to facilitate orientation for embedding, the peritoneal cavity was opened at the caudal end and a small piece of cardboard was inserted beneath the peritoneum. The anterior abdominal wall adhered to the cardboard and was excised. Tissues were fixed and embedded in paraffin (Sainte-Marie, 1962). Serial sections were cut at a thickness of 6 M from multiple areas of the wound and stained by the indirect method for immunofluorescent microscopy (Coons, 1958). Adjacent serial sections were reacted with epithelial specific rabbit antibasement membrane (anti-EBM) (Pierce et al., 1964) or in the case of the control with normal rabbit serum. Anti-EBM reacts only with the basement membranes of epithelial cells, and does not react with those of endothelium or muscle cells or with collagen. The sections were then stained with fluorescein isothiocyanate-labeled sheep antirabbit y-globulin and examined in a Leitz fluorescent microscope
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JOHNSON
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equipped with a Polaroid camera. Photographs were taken with exposure times varying from 10 to 60 seconds depending on the intensity of fluorescence. Autofluorescence of epidermis was always a major problem, particularly when recording results in “black and white” prints. Consequently, photographs were routinely underexposed, which reduced the autofluorescence in relationship to the specific, but it also made orientation of specimens more difficult. To ensure proper orientation of specimens, camera lucida drawings were made (Figs. la, 2a, 3a, 4a). To obtain optimal fixation for electron microscopy, the wound and adjacent skin were infiltrated with Verona1 acetate buffered 0~01, pH 7.2 (Palade, 1952) by subcutaneous injection at the time of sacrifice. When the injected area was uniformly dark brown to black, the wound and adjacent 2 to 3 mm of normal skin were excised and immersed in buffered 0~04 to complete fixation. The blocks of tissue were embedded in an Epon:Araldite mixture and thick- and thinsectioned using an LKB Ultrotome. To ensure proper orientation, 1 p-thick sections were examined and photographed in a Zeiss Photomicroscope II using phase optics. Adjacent silver or gold thin sections on coated grids were stained with uranyl acetate and examined in a Philips 200 electron microscope. RESULTS
Reaction of anti-EBM with skin resulted in a continuous narrow band of bright specific fluorescence between epidermis and dermis corresponding to the anatomical location of the basement membrane. In addition, it reacted with basement membranes of the glomerulus and anterior lens capsule, reactions that could be abolished by absorbing the antiserum with glomerular basement membrane, or lens capsule. Autofluorescence was minimal in the dermis and always marked in the overlying epidermis. The dermis, in confirmation of previous experience, never contained epithelial basement membrane antigen. Neither endothelial basement membrane, collagen, nor other component of the connective tissues reacted with anti-EBM. Adjacent serial sections reacted with normal rabbit serum had only nonspecific autofluorescence in the epidermis. Epithelial basement membrane antigen always extended to the wound margin in all specimens examined at 0 and 1 hours (Fig. la, b, and c), but by 2 hours the antigen had disappeared for a distance of
BASEMENT
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HEALING
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2 to 4 cell diameters from the margin of the wound. This was an invariable finding. Elsewhere the basement membrane antigen fluoresced as usual. Although leukocytes had migrated into the area, there was no detectable fluorescence in them as might be expected if they had phagocytized the basement membrane. After the disappearance of epithelial basement membrane antigen from the margin of the wound, migration and proliferation of epithelial cells began, as reported by others (Winter, 1964; Odland and Ross, 1963). Closure was complete between 43 and 72 hours after wounding, leaving a thick mass of redundant epithelium which extended deeply into the corium and bulged over the surface of the skin. Hyperkeratosis was usual over this redundant epithelium. During the examination of 1 p thick sections of plastic embedded material for the orientation of sections for electron microscopy, dark, irregular cells were observed in the basal layer of the epithelium at the time of complete reepithelialization, and during remodeling of the wound. These cells, which constituted approximately one half of the basal cell population, appeared to be degenerating. They were small with irregular borders, pyknotic or no nucleus, and scant granular cytoplasm (Fig. 5). Cells of similar appearance were rarely if ever seen in the spinous or granular layers. The adjacent, uninjured epithelium at 72 hours and the epithelium at other times during would healing were devoid of these cells, suggesting that they were degenerate and not an artifact of fixation. Remodeling of the wound began shortly after the completion of reepithelialization and continued for the next 7 to 10 days, depending on the width of the original defect. At the end of this process, the epithelium covering the wound could be distinguished from adjacent, uninjured areas only by a slightly thickened keratotic layer. Epithelial basement membrane antigen was not present beneath the migrating and proliferating epithelial cells which closed the wound during the first 72 hours (Fig. 3a, b, c). The epithelial basement membrane antigen terminated abruptly near the original margin of the wound and did not accompany or precede the epithelial cells which closed it. The antigen was absent beneath the large redundant epithelial mass that protruded into the corium and reappeared only after remodeling was complete at about 7-10 days after wounding. No specific fluorescence was detected within the epiderma1 cells during this phase.
.-. Figures la, 2a, 3a and 4a are camera lucida drawings of sections of skin shown in the accompanying fluorescent micrographs. Note the location of specific fluorescence in the basement membrane (heavy line). Arrows denote the end of the basement membrane antigen nearest the wound margin. The areas enclosed in boxes represent the tissue illustrated in the electron micrographs. Figures la through 4c are x 366. FIG. 1. Serial sections of a wound at 0 hour stained with fluorescein-labeled antiepithelial basement membrane (b) or normal rabbit serum (c). The basement mem-
BASEMENT
Electron
MEMBRANE
IN WOUND
HEALING
539
Microscopy
The ultrastructure of the basement membrane was followed chronologically and particular attention was paid to correlating fine structural detail with the changes in antigenicity of the basement membrane, and the degenerating cells observed during remodeling. The margin of the wound was obliterated by debris at 0 hours, yet the basement membrane was clearly defined and extended to the margin (Fig. 6). It had an amorphous or finely fibrillar texture and consisted of distinct laminae densa and lucida of approximately equal widths. The plasma membranes of the marginal epithelial cells were distinct at the surface but were incorporated in the debris at the base of the wound. The degenerated state of marginal cells, presumably the result of mechanical trauma, was apparent even though the animals had been sacrificed shortly after wounding and the tissue promptly fixed. The mitochondria in particular demonstrated the effects of injury as was apparent by the dense matrices which contrasted with dilated, electron lucent spaces between the cristae (Fig. 6). The loss of antigenicity of basement membrane beneath the injured cells of the wound margin at 2 hours was accompanied by a less striking yet distinct ultrastructural alteration, which we would have overlooked had the immunological study not directed our attention to it. The laminae densa and lucida, which normally identified the basement membrane, no longer existed as clearly defined lamellae. Instead, the lamina lucida was obliterated in a patchy manner by a material the texture of which differed slightly from the usual brane separates the epidermis and dermis and extends to the wound margin (arrow in a and b). A small amount of autofluorescence is present in the epidermis. The control, c, is negative. FIG. 2. Serial sections of a wound at 2 hours stained with fluorescein-labeled antiepithelial basement membrane (b) or normal rabbit serum (c). The basement membrane, present beneath the normal epidermis, has disappeared abruptly beneath the marginal 4-10 cells (arrow in a and b). The wound margin is seen in the lower right comer. FIG. 3. Serial sections of a wound stained at 24 hours with fluorescein-labeled antiepithelial basement membrane (b) or normal rabbit serum (c). The basement membrane is absent beneath the wound epithelium and ends abruptly at the point where normal and redundant wound epithelium meet (arrow in a and b). FIG. 4. Serial sections of a wound stained at 72 hours with fluorescein-labeled antiepithelial basement membrane (b) or normal rabbit serum (c). The basement membrane abruptly ends at the edges of the redundant epidermis which closed the wound (arrows in a and b). The large scab (S) shows a small amount of autofluorescence.
540
JOHNSON
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PIERCE
FIG. 5. Phase micrograph redundant generating
of a wound at 72 hours illustrating the thickness of the epithelium (6 cells in relationship to 2 to 3 cells normally) and the dark decells (arrows) which are confined to the basal layer. X 1400.
lamina densa. The cells lying on this altered basement membrane were characterized by enlarged, vacuolated mitochondria with disrupted cristae and loss of hemidesmosomes (Fig. 7). The basement membrane beneath the fifth and all subsequent cells lateral from the margin, which as noted previously contained antigen, was unaltered as was the ultrastructural appearance of the cells. At 24 hours, a broad layer of amorphous material was present beneath the marginal migrating epidermal cells which contained very little fibrin in contrast to other reports (Odland and Boss, 1966). The material beneath the remainder of the migrating cells was finely fibrillar, extended several microns into the dermis and ap eared to be degenerating collagen. A few ill-defined fibers with 640 R periodicity were seen in it (Fig. 8). Since this material failed to react immunohistochemically with anti-EBM we presume it to be a mixture of degenerating collagen plus serous exudate. The migrating epidermal cells in the basal layer contained numerous poorly defined electron dense regions of the plasma membrane which appeared to be newly forming hemidesmosomesin direct contact with the substratum.
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MEMBRANE
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FIG. 6. Electron micrograph illustrating the margin of a wound at 0 hours. The basement membrane (bm) extends to the wound margin (arrow at left). The plasma membrane (pm) of the marginal epidermal cell is distinct but becomes obliterated by debris (de) near the basement membrane. The mitochondria (m) are abnormal in appearance. The membranes of cristae are widened, blurred, and separated from each other by an electron lucent material which contrasts with the dense matrix and imparts an appearance akin to negative staining. The dermis is composed of well oriented bundles of collagen fibers. X 25,000.
Figure 9 is a representative electron micrograph of wounds examined at the time of closure. Although basement membrane antigen could not be demonstrated by immunofluorescence, beneath the epithelium covering the wound an apparently normal basement membrane was present. Between normal appearing basal cells and comprising about onehalf of the basal cell population were the dark, irregular, degenerating cells observed by phase microscopy in the adjacent serial sections. These cells were characterized by pyknotic nuclei, disrupted rough endoplasmic reticulum, clumped membranes and numerous free ribosomes, and vacuolated, swollen mitochondria with few cristae. This type of degeneration has been described in healing human skin (Odland and Ross, 1968), but not to the extent seen in murine skin.
FIG. 7. Electron micrograph from a wound at 2 hours (as noted in Fig. 2a). Material resembling the lamina densa of the basement membrane (arrows) completely fills the space between the dermis and epidermis and obliterates the lamina lucida. Note the enlarged, vacuolated mitochondria (m) with disrupted cristae. Occasional, poorly defined hemidesmosomes can be seen (h). The bundles of collagen in the dermis appear loosened. Compare to appearance of basement membrane beneath normal epidermis (Fig. 10). X 23,500. 542
BASEMENT
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IN WOUND
HEALING
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FIG. 8. Electron micrograph from a wound at 24 hours as noted in Fig. 3a. A hand of finely fibrillar material extends for several microns into the dermis and contains occasional membrane bound vacuoles (u). A fragment of a collagen fiber with blurred cross striations is present (c), suggesting that the fine fibrils are degraded collagen or have engulfed collagen. The thickened areas of the plasma membrane at the right are probably poorly defined, newly forming hemidesmosomes (h). X 39,199.
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JOHNSON
AND
PIERCE
FIG. 9. Electron micrograph from a wound at 72 hours showing basement membrane (bm) beneath three cells in the area of redundant epithelium denoted in Fig. 4a (center box). The basement membrane (bm) is composed of distinct lamina densa and lucida of equal width. The degenerated cell in the center (dc) contains vacuolated mitochondria (m) with disrupted cristae (compare with the mitochondria in the adjacent cells), loss of rough endoplasmic reticulum and clumps of free ribosomes. The cells on either side of the degenerating one are normal in appearance (hc) and are an intrinsic control suggesting that the cell, dc, is truly degenerating. Compare with Fig. 10, which is from the same block of tissue. x 14,000.
-
--
FIG. 10. Electron
---
-.--
.-I-
-._. --..
---
micrograph of normal skin (area shown in the lateral box in Fig. 4a). Note the distinct lamina densa and lucida of the basement membrane (bm). The basal epidermal cell demonstrates adequate fixation with intact mitochondria (m) containing delicate cristae. Numerous vertically oriented tonofibrils (t) are present. Desmosomes (d) are present along the plasma membrane between two basal cells, and numerous hemidesmosomes (h) are in apposition to the basement membrane. The dermis contains several collagen fibers with periodicity. X 23,500.
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The appearance of these altered basal cells could be attributed to either degeneration or an artifact of fixation. Artifacts due to improper fixation were excluded by the excellence of preservation and appearance of basal cells adjacent to the degenerated cells, as demonstrated in Fig. 9, and also by the appearance of the uninvolved basal layer lateral to the wound which was routinely examined as a further control (Fig. 10). Once reepithelialization was complete, the wound epithelium entered the remodeling phase, during which the thickened epithelial layer progressively thinned and was finally indistinguishable from adjacent, unaltered epidermis. Although the epithelium regained all normal characteristics, the area was easily distinguished by the disorientation of the dermal collagen fibers. The basement membrane consisted of well-defined laminae densa and lucida and was continuous beneath the epidermis. DISCUSSION
The most important observation from this study was the loss of antigenicity of epithelial basement membrane which correlated with a change in the ultrastructural appearance of the membrane immediately prior to the migration and proliferation of epidermal cells in the closure of the wound. It is probable that the changes in basement membrane are the result of the action of the “collagenase” which Grill0 and Gross (1967) discovered in guinea pig skin. This collagenase is synthesized by the epithelium at the wound margin, and it cleaves fibrillar collagen into two portions, one containing three quarters of the amino acids and the other one quarter (Kang et al., 1966). After this initial cleavage, the collagen molecule becomes susceptible to further degradation by a spectrum of proteolytic enzymes. Epithelial basement membrane contains collagen (Kefalides, 1968; Spiro, 1967), so it is not improbable that the enzyme might degrade it. Since the epithelial-specific antigen of basement membrane is believed to be in the noncollagenous portion of the molecule, the collagenase may also degrade the noncollagenous part or render it susceptible to degradation by other enzymes. These changes in epithelial basement membrane preceded migration and proliferation of epithelial cells, and raise questions concerning the relationship of these events. Although they may be unrelated, evidence from other experiments suggests that they may be causally related. When epithelial cells are injured by physical,
BASEMENT
MEMBRANE
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chemical, or microbial agents, they synthesize excessive amounts of basement membrane, presumably to protect themselves from a hostile environment (Pierce and Nakane, 1969). This suggests that epithelial basement membrane may play a role in maintaining optimal environmental conditions for epithelial cells. Wessells (1963) has shown that dermal factors are necessary for mitosis to occur in epidermal cells. Thus one might postulate that in the normal situation dermal factors traverse basement membrane and play a role in regulating the proliferation of basal cells to renew epithelium. After wounding, with loss of antigenicity and a change in the ultrastructural appearance of a portion of the basement membrane adjacent to the margin of the wound, cells would be mechanically released from their attachments to basement membrane as suggested by changes observed in the hemidesmosomes. This would allow for migration and the changes in basement membrane could allow for freer access of the dennal factors as described by Wessells, thereby promoting cell division. Two observations worthy of comment occurred during remodeling of the redundant epithelium of the wound. A peculiar alteration in the appearance of approximately half of the cells of the basal layer was observed with phase and electron microscopes. Similar appearances were observed when wounds were fixed in osmic acid, glutaraldehyde or paraformaldehyde and when the osmolarities of these fixatives were varied. In addition, cells of this appearance were not present in the uninvolved basal layer of normal skin from the same block of tissue. Consequently, we believe the ultrastructural appearance is not due to an artifact of fixation, and represents a type of degeneration. Whether or not this type of degeneration is lethal to the cells remains to be seen, but its presence would certainly suggest either reduced metabolic activity or selective death of basal cells as a mechanism of remodeling redundant wound epithelium. Fallon and Saunders (1968) described selective cell death as a mechanism for remodeling of the digits during wing bud development in the chick embryo. Thus there is a precedent for this postulated mechanism for remodeling of wound epithelium. It is of interest that Ross (1970) has observed similar cells in his ultrastructural studies of wound healing. The second noteworthy observation was that although epithelial basement membrane antigen was absent during remodeling, an ultrastructurally unaltered basement membrane was present beneath the
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remodeled epithelium suggesting that a less extensive hydrolysis of the basement membrane was occurring. The inability to detect antigen cannot be due to lack of sensitivity of the immunohistochemical assay. Pierce et al. (1962) have demonstrated the presence of specific fluorescence within the cytoplasm of parietal yolk sac carcinoma cells in a pattern that correlated in distribution and amount with that of the endoplasmic reticulum. Thus very small amounts of antigen can be detected. Since degenerating epithelial cells of wounds (Grill0 and Gross, 1967) and of the gut of tadpole (Gross and Lapiere, 1962) hydrolyze collagen, it is probable that these degenerating cells also synthesize proteolytic enzymes that cause basement membrane to be partially hydrolyzed, and it is conceivable that antigenicity might be lost and definable ultrastructure remain. In support of this idea is the work of Michaeli et al. (1969), for example, which shows that antigenic determinants can be split from the collagen molecule without destroying the helical structure. SUMMARY
The healing of cutaneous wounds of mice was examined by immunofluorescent, phase, and electron microscopy. The results suggested that the epithelial basement membrane appeared to undergo degradation early in the healing process and did not regain all characteristics until remodeling of the redundant epidermis was completed. It did not appear to function as a substratum for epithelial migration; instead it appeared that the basement membrane may be partially responsible for the maintenance of the microenvironment of the epidermis. Phase micrographs illustrated a large number of degenerating basal cells at the completion of reepithelialization. These cells were confined to the basal layer and suggested that wound remodeling was initiated by selective cell death. These studies were supported by grants GM977 and AM13112 from Institutes of Health. The technical assistance of Mr. Marlin Nofziger, Jones and Mr. Howard Mitchell was deeply appreciated.
the National Mr. Alan S.
REFERENCES COONS, A. H. (1958). Fluorescent methods. In “General Cytochemical Methods” Danielli, ed.), pp. 399-435. Academic Press, New York. FALLON, J. F., and SAUNDERS, J. W., JR. (1968). In vitro analysis of the control death in the zone of prospective necrosis from the chick wing bud. Develop. 18, 533-570.
(J. F. of cell Biol.
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GIACOMETTI, L., and PARAKKAL, P. F. (1969). Skin transplantation: Orientation of epithelial cells by the basement membrane. Nature (London) 223,514-515. GRILLO, H. C., and GROSS, J. (1967). Collagenolytic activity during wound repair. Deoelop. Biol. 15, 300-317. GROSS, J., and LAPIERE, C. M. (1962). Collagenolytic activity in amphibian tissues: A tissue culture assay. Boc. Nat. Acad. Sci. U. S. 48,1014-1022. KANG, A. H., NAGAI, Y., PIEZ, K. A., and GROSS, J. (1966). Studies on the structure of collagen utilizing a collagenolytic enzyme from tadpoles. Biochemistry 5, 509-515. KEFALIDES, N. A. (1969). Isolation and characterization of the collagen from glomerular basement membrane. Biochemistry 7, 3103-3112. MUKERJEE, H., SRI RAM, J., and PIERCE, G. B., JR. (1965). Basement membranes. V. Chemical composition of neoplastic basement membrane mucoprotein. Amer. J. Pathol. 46, 49-57. MICHAELI, D., MARTIN, G. R., KFITMAN, J., BENJAMIN, E., LEUNG, D. Y. K., and BLAT, B. A. (1969). Localization of antigenic determinants in the polypeptide chains of collagen. Science 166, 1522-1523. MIDGLEY, A. R., JR., and PIERCE, G. B., JR. (1963). Immunohistochemical analysis of basement membranes of the mouse. Amer. J. Fbthol. 43,929-943. ODLAND, G., and ROSS, R. (1966). Human wound repair. I. Epidermal regeneration. J.
Cell Biol. 39, 135-151. PALADE,
G. E. (1952).
A study
of fiiation
for electron
microscopy.
J. Erp. Med. 95,
285-298. PIERCE, G. B., and NAKANE, P. K. (1969). Basement membranes. Synthesis and deposition in response to cellular injury. Lab. Znuest. 21,27-41. PIERCE, G. B., JR., MIDGLEY, A. R., JR., SRI RAM, J., and FELDMAN, J. D. (1962). Parietal yolk sac carcinoma: Clue to the histogenesis of Reichert’s membrane of the mouse embryo. Amer. J. Puthol. 41, 549-566. PIERCE, G. B., JR., BEALS, T. F., SRI RAM, J., and MIDGLEY, A. R., JR. (1964). Basement membranes. IV. Epithelial origin and immunologic cro8s reactions. Amer. J. Pathol. 45, 929-961. Ross, R. (1970). Personal communication. SAINTE-MARIE, G. (1962). A paraffin embedding technique for studies employing immunofluorescence. J. Histochem. Cytochem. 10, 250-256. SCH~LLING, J. A. (1968). Wound healing. Physiol. Rev. 48,374423. SCHMIDT, A. J. (1966). “Cellular Biology of Vertebrate Regeneration and Repair,” pp. 10-16. Univ. of Chicago Press, Chicago, Illinois. SPIRO, R. G. (1967). Studies on the renal glomerular basement membrane. Nature of the carbohydrate units and their attachment to the peptide portion. J. Biol. Chem. 242,1923-1932. SPIRO, R. G., and FUKUSHI, S. (1969). The lens capsule: Studies on the carbohydrate units. J. Biol. Chem. 244, 2049-2058. WESSELLS, N. K. (1963). Effects of extra-epithelial factors on the incorporation of thymidine by embryonic epidermis. Exp. Cell Res. 30,36-55. WINTER, G. D. (1964). Movement of epidermal cells over the wound surface. Aduan. Biol. Skin 5, 113-127.
BIOLOGY
Changes
23, 534-5&l
(1970)
in Antigenicity of Basement during Wound Healing
Membrane
LEWIS D. JOHNSON AND G. BARRY PIERCE Department
of Pathology,
Denver, Accepted
University of Colorado Colorado 80220 September
Medical
Center,
10, 1970
INTRODUCTION
Through the isolation and manipulation of epithelial tumors in vitro, it has been possible to demonstrate by direct means that epithelial cells synthesize the basement membranes upon which they rest. These have been named epithelial basement membranes’ to distinguish them from those of endothelium and muscle cells, for example. Chemical analyses of epithelial basement membrane produced in vitro or in the ascites (Mukerjee et al., 1965), situations lacking contamination by connective tissues, have confirmed the idea that there was a tropocollagen core in the molecule. To this collagen is linked a mucoprotein (Kefalides, 1968; Spiro, 1967; Spiro and Fukushi, 1969) which is antigenic and serves as a specific marker for all epithelial basement membranes of a particular species (Midgley and Pierce, 1963; Pierce et al., 1964). Physical, chemical, or microbial injury of epithelial cells results in increased synthesis of epithelial basement membrane, an observation compatible with the notion that basement membrane may subserve a protective role for the adjacent cells (Pierce and Nakane, 1969). Little is known about the turnover of basement membrane, although Grill0 and Gross (1967) have demonstrated an enzyme synthesized by epithelial cells at the margins of cutaneous wounds which cleaves the collagen molecule at a specific place, and which has been named “collagenase”. In view of its collagenous component it is conceivable that one of the substrates of this enzyme may be basement membrane. If this were the case, one might expect enzymatic degradation of epithelial basement membrane during the healing of cutaneous wounds. 1 Epithelial basement membrane called basal lamina or adepidermal basement membrane of amphibians, layers of collagen.
applies to what, in the mammal has also been membrane, and it is not to be confused with the which in addition, includes several well ordered 534
BASEMENT
MEMBRANE
IN
WOUND
HEALING
535
A great deal has been learned about wound healing including studies of the mitotic activity and migration of epithelial cells, and the synthesis of collagen by fibroblasts to restore the epithelium and dermis, respectively (reviews by Schilling, 1968; Schmidt, 1968). Despite the extent of the studies, only brief reference has ever been made to epithelial basement membrane during wound healing (Odland and Ross, 1968; Giacometti and Parakkal, 1969). In a study to be reported here, we have examined wounds timewise, paying particular attention to the antigenicity and ultrastructure of the epithelial basement membrane. Epithelial basement membrane antigen disappeared from the wound margin 2 hours after wounding, then epithelium migrated across a substratum of fibrin, serum proteins, and collagen to “close” the wound, and the epithelial basement membrane antigen did not reappear until after the completion of remodeling of the wound epithelium. MATERIALS
AND
METHODS
Male ICR mice, 6-8 weeks old, were prepared by shaving the abdominal wall, and longitudinal incisions were made through all skin layers down to the fascia. Most animals were incised in three places, to ensure that adequate material could be taken from each mouse for both immunochemistry and electron microscopy. Each incision was approximately 2 cm in length and gaped 2 to 3 mm at the widest point. Bleeding was minimal and although sterile procedure was not followed, infected wounds were infrequent and were not examined. The wounds were allowed to heal without closure or dressings. Mice were sacrificed at 0, 1, 2, 4, 8, 16, and 24 hours, then at 24-hour intervals through the tenth day. To prevent curling of the specimen and to facilitate orientation for embedding, the peritoneal cavity was opened at the caudal end and a small piece of cardboard was inserted beneath the peritoneum. The anterior abdominal wall adhered to the cardboard and was excised. Tissues were fixed and embedded in paraffin (Sainte-Marie, 1962). Serial sections were cut at a thickness of 6 M from multiple areas of the wound and stained by the indirect method for immunofluorescent microscopy (Coons, 1958). Adjacent serial sections were reacted with epithelial specific rabbit antibasement membrane (anti-EBM) (Pierce et al., 1964) or in the case of the control with normal rabbit serum. Anti-EBM reacts only with the basement membranes of epithelial cells, and does not react with those of endothelium or muscle cells or with collagen. The sections were then stained with fluorescein isothiocyanate-labeled sheep antirabbit y-globulin and examined in a Leitz fluorescent microscope
536
JOHNSON
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PIERCE
equipped with a Polaroid camera. Photographs were taken with exposure times varying from 10 to 60 seconds depending on the intensity of fluorescence. Autofluorescence of epidermis was always a major problem, particularly when recording results in “black and white” prints. Consequently, photographs were routinely underexposed, which reduced the autofluorescence in relationship to the specific, but it also made orientation of specimens more difficult. To ensure proper orientation of specimens, camera lucida drawings were made (Figs. la, 2a, 3a, 4a). To obtain optimal fixation for electron microscopy, the wound and adjacent skin were infiltrated with Verona1 acetate buffered 0~01, pH 7.2 (Palade, 1952) by subcutaneous injection at the time of sacrifice. When the injected area was uniformly dark brown to black, the wound and adjacent 2 to 3 mm of normal skin were excised and immersed in buffered 0~04 to complete fixation. The blocks of tissue were embedded in an Epon:Araldite mixture and thick- and thinsectioned using an LKB Ultrotome. To ensure proper orientation, 1 p-thick sections were examined and photographed in a Zeiss Photomicroscope II using phase optics. Adjacent silver or gold thin sections on coated grids were stained with uranyl acetate and examined in a Philips 200 electron microscope. RESULTS
Reaction of anti-EBM with skin resulted in a continuous narrow band of bright specific fluorescence between epidermis and dermis corresponding to the anatomical location of the basement membrane. In addition, it reacted with basement membranes of the glomerulus and anterior lens capsule, reactions that could be abolished by absorbing the antiserum with glomerular basement membrane, or lens capsule. Autofluorescence was minimal in the dermis and always marked in the overlying epidermis. The dermis, in confirmation of previous experience, never contained epithelial basement membrane antigen. Neither endothelial basement membrane, collagen, nor other component of the connective tissues reacted with anti-EBM. Adjacent serial sections reacted with normal rabbit serum had only nonspecific autofluorescence in the epidermis. Epithelial basement membrane antigen always extended to the wound margin in all specimens examined at 0 and 1 hours (Fig. la, b, and c), but by 2 hours the antigen had disappeared for a distance of
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2 to 4 cell diameters from the margin of the wound. This was an invariable finding. Elsewhere the basement membrane antigen fluoresced as usual. Although leukocytes had migrated into the area, there was no detectable fluorescence in them as might be expected if they had phagocytized the basement membrane. After the disappearance of epithelial basement membrane antigen from the margin of the wound, migration and proliferation of epithelial cells began, as reported by others (Winter, 1964; Odland and Ross, 1963). Closure was complete between 43 and 72 hours after wounding, leaving a thick mass of redundant epithelium which extended deeply into the corium and bulged over the surface of the skin. Hyperkeratosis was usual over this redundant epithelium. During the examination of 1 p thick sections of plastic embedded material for the orientation of sections for electron microscopy, dark, irregular cells were observed in the basal layer of the epithelium at the time of complete reepithelialization, and during remodeling of the wound. These cells, which constituted approximately one half of the basal cell population, appeared to be degenerating. They were small with irregular borders, pyknotic or no nucleus, and scant granular cytoplasm (Fig. 5). Cells of similar appearance were rarely if ever seen in the spinous or granular layers. The adjacent, uninjured epithelium at 72 hours and the epithelium at other times during would healing were devoid of these cells, suggesting that they were degenerate and not an artifact of fixation. Remodeling of the wound began shortly after the completion of reepithelialization and continued for the next 7 to 10 days, depending on the width of the original defect. At the end of this process, the epithelium covering the wound could be distinguished from adjacent, uninjured areas only by a slightly thickened keratotic layer. Epithelial basement membrane antigen was not present beneath the migrating and proliferating epithelial cells which closed the wound during the first 72 hours (Fig. 3a, b, c). The epithelial basement membrane antigen terminated abruptly near the original margin of the wound and did not accompany or precede the epithelial cells which closed it. The antigen was absent beneath the large redundant epithelial mass that protruded into the corium and reappeared only after remodeling was complete at about 7-10 days after wounding. No specific fluorescence was detected within the epiderma1 cells during this phase.
.-. Figures la, 2a, 3a and 4a are camera lucida drawings of sections of skin shown in the accompanying fluorescent micrographs. Note the location of specific fluorescence in the basement membrane (heavy line). Arrows denote the end of the basement membrane antigen nearest the wound margin. The areas enclosed in boxes represent the tissue illustrated in the electron micrographs. Figures la through 4c are x 366. FIG. 1. Serial sections of a wound at 0 hour stained with fluorescein-labeled antiepithelial basement membrane (b) or normal rabbit serum (c). The basement mem-
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Microscopy
The ultrastructure of the basement membrane was followed chronologically and particular attention was paid to correlating fine structural detail with the changes in antigenicity of the basement membrane, and the degenerating cells observed during remodeling. The margin of the wound was obliterated by debris at 0 hours, yet the basement membrane was clearly defined and extended to the margin (Fig. 6). It had an amorphous or finely fibrillar texture and consisted of distinct laminae densa and lucida of approximately equal widths. The plasma membranes of the marginal epithelial cells were distinct at the surface but were incorporated in the debris at the base of the wound. The degenerated state of marginal cells, presumably the result of mechanical trauma, was apparent even though the animals had been sacrificed shortly after wounding and the tissue promptly fixed. The mitochondria in particular demonstrated the effects of injury as was apparent by the dense matrices which contrasted with dilated, electron lucent spaces between the cristae (Fig. 6). The loss of antigenicity of basement membrane beneath the injured cells of the wound margin at 2 hours was accompanied by a less striking yet distinct ultrastructural alteration, which we would have overlooked had the immunological study not directed our attention to it. The laminae densa and lucida, which normally identified the basement membrane, no longer existed as clearly defined lamellae. Instead, the lamina lucida was obliterated in a patchy manner by a material the texture of which differed slightly from the usual brane separates the epidermis and dermis and extends to the wound margin (arrow in a and b). A small amount of autofluorescence is present in the epidermis. The control, c, is negative. FIG. 2. Serial sections of a wound at 2 hours stained with fluorescein-labeled antiepithelial basement membrane (b) or normal rabbit serum (c). The basement membrane, present beneath the normal epidermis, has disappeared abruptly beneath the marginal 4-10 cells (arrow in a and b). The wound margin is seen in the lower right comer. FIG. 3. Serial sections of a wound stained at 24 hours with fluorescein-labeled antiepithelial basement membrane (b) or normal rabbit serum (c). The basement membrane is absent beneath the wound epithelium and ends abruptly at the point where normal and redundant wound epithelium meet (arrow in a and b). FIG. 4. Serial sections of a wound stained at 72 hours with fluorescein-labeled antiepithelial basement membrane (b) or normal rabbit serum (c). The basement membrane abruptly ends at the edges of the redundant epidermis which closed the wound (arrows in a and b). The large scab (S) shows a small amount of autofluorescence.
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FIG. 5. Phase micrograph redundant generating
of a wound at 72 hours illustrating the thickness of the epithelium (6 cells in relationship to 2 to 3 cells normally) and the dark decells (arrows) which are confined to the basal layer. X 1400.
lamina densa. The cells lying on this altered basement membrane were characterized by enlarged, vacuolated mitochondria with disrupted cristae and loss of hemidesmosomes (Fig. 7). The basement membrane beneath the fifth and all subsequent cells lateral from the margin, which as noted previously contained antigen, was unaltered as was the ultrastructural appearance of the cells. At 24 hours, a broad layer of amorphous material was present beneath the marginal migrating epidermal cells which contained very little fibrin in contrast to other reports (Odland and Boss, 1966). The material beneath the remainder of the migrating cells was finely fibrillar, extended several microns into the dermis and ap eared to be degenerating collagen. A few ill-defined fibers with 640 R periodicity were seen in it (Fig. 8). Since this material failed to react immunohistochemically with anti-EBM we presume it to be a mixture of degenerating collagen plus serous exudate. The migrating epidermal cells in the basal layer contained numerous poorly defined electron dense regions of the plasma membrane which appeared to be newly forming hemidesmosomesin direct contact with the substratum.
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FIG. 6. Electron micrograph illustrating the margin of a wound at 0 hours. The basement membrane (bm) extends to the wound margin (arrow at left). The plasma membrane (pm) of the marginal epidermal cell is distinct but becomes obliterated by debris (de) near the basement membrane. The mitochondria (m) are abnormal in appearance. The membranes of cristae are widened, blurred, and separated from each other by an electron lucent material which contrasts with the dense matrix and imparts an appearance akin to negative staining. The dermis is composed of well oriented bundles of collagen fibers. X 25,000.
Figure 9 is a representative electron micrograph of wounds examined at the time of closure. Although basement membrane antigen could not be demonstrated by immunofluorescence, beneath the epithelium covering the wound an apparently normal basement membrane was present. Between normal appearing basal cells and comprising about onehalf of the basal cell population were the dark, irregular, degenerating cells observed by phase microscopy in the adjacent serial sections. These cells were characterized by pyknotic nuclei, disrupted rough endoplasmic reticulum, clumped membranes and numerous free ribosomes, and vacuolated, swollen mitochondria with few cristae. This type of degeneration has been described in healing human skin (Odland and Ross, 1968), but not to the extent seen in murine skin.
FIG. 7. Electron micrograph from a wound at 2 hours (as noted in Fig. 2a). Material resembling the lamina densa of the basement membrane (arrows) completely fills the space between the dermis and epidermis and obliterates the lamina lucida. Note the enlarged, vacuolated mitochondria (m) with disrupted cristae. Occasional, poorly defined hemidesmosomes can be seen (h). The bundles of collagen in the dermis appear loosened. Compare to appearance of basement membrane beneath normal epidermis (Fig. 10). X 23,500. 542
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FIG. 8. Electron micrograph from a wound at 24 hours as noted in Fig. 3a. A hand of finely fibrillar material extends for several microns into the dermis and contains occasional membrane bound vacuoles (u). A fragment of a collagen fiber with blurred cross striations is present (c), suggesting that the fine fibrils are degraded collagen or have engulfed collagen. The thickened areas of the plasma membrane at the right are probably poorly defined, newly forming hemidesmosomes (h). X 39,199.
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FIG. 9. Electron micrograph from a wound at 72 hours showing basement membrane (bm) beneath three cells in the area of redundant epithelium denoted in Fig. 4a (center box). The basement membrane (bm) is composed of distinct lamina densa and lucida of equal width. The degenerated cell in the center (dc) contains vacuolated mitochondria (m) with disrupted cristae (compare with the mitochondria in the adjacent cells), loss of rough endoplasmic reticulum and clumps of free ribosomes. The cells on either side of the degenerating one are normal in appearance (hc) and are an intrinsic control suggesting that the cell, dc, is truly degenerating. Compare with Fig. 10, which is from the same block of tissue. x 14,000.
-
--
FIG. 10. Electron
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-.--
.-I-
-._. --..
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micrograph of normal skin (area shown in the lateral box in Fig. 4a). Note the distinct lamina densa and lucida of the basement membrane (bm). The basal epidermal cell demonstrates adequate fixation with intact mitochondria (m) containing delicate cristae. Numerous vertically oriented tonofibrils (t) are present. Desmosomes (d) are present along the plasma membrane between two basal cells, and numerous hemidesmosomes (h) are in apposition to the basement membrane. The dermis contains several collagen fibers with periodicity. X 23,500.
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The appearance of these altered basal cells could be attributed to either degeneration or an artifact of fixation. Artifacts due to improper fixation were excluded by the excellence of preservation and appearance of basal cells adjacent to the degenerated cells, as demonstrated in Fig. 9, and also by the appearance of the uninvolved basal layer lateral to the wound which was routinely examined as a further control (Fig. 10). Once reepithelialization was complete, the wound epithelium entered the remodeling phase, during which the thickened epithelial layer progressively thinned and was finally indistinguishable from adjacent, unaltered epidermis. Although the epithelium regained all normal characteristics, the area was easily distinguished by the disorientation of the dermal collagen fibers. The basement membrane consisted of well-defined laminae densa and lucida and was continuous beneath the epidermis. DISCUSSION
The most important observation from this study was the loss of antigenicity of epithelial basement membrane which correlated with a change in the ultrastructural appearance of the membrane immediately prior to the migration and proliferation of epidermal cells in the closure of the wound. It is probable that the changes in basement membrane are the result of the action of the “collagenase” which Grill0 and Gross (1967) discovered in guinea pig skin. This collagenase is synthesized by the epithelium at the wound margin, and it cleaves fibrillar collagen into two portions, one containing three quarters of the amino acids and the other one quarter (Kang et al., 1966). After this initial cleavage, the collagen molecule becomes susceptible to further degradation by a spectrum of proteolytic enzymes. Epithelial basement membrane contains collagen (Kefalides, 1968; Spiro, 1967), so it is not improbable that the enzyme might degrade it. Since the epithelial-specific antigen of basement membrane is believed to be in the noncollagenous portion of the molecule, the collagenase may also degrade the noncollagenous part or render it susceptible to degradation by other enzymes. These changes in epithelial basement membrane preceded migration and proliferation of epithelial cells, and raise questions concerning the relationship of these events. Although they may be unrelated, evidence from other experiments suggests that they may be causally related. When epithelial cells are injured by physical,
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chemical, or microbial agents, they synthesize excessive amounts of basement membrane, presumably to protect themselves from a hostile environment (Pierce and Nakane, 1969). This suggests that epithelial basement membrane may play a role in maintaining optimal environmental conditions for epithelial cells. Wessells (1963) has shown that dermal factors are necessary for mitosis to occur in epidermal cells. Thus one might postulate that in the normal situation dermal factors traverse basement membrane and play a role in regulating the proliferation of basal cells to renew epithelium. After wounding, with loss of antigenicity and a change in the ultrastructural appearance of a portion of the basement membrane adjacent to the margin of the wound, cells would be mechanically released from their attachments to basement membrane as suggested by changes observed in the hemidesmosomes. This would allow for migration and the changes in basement membrane could allow for freer access of the dennal factors as described by Wessells, thereby promoting cell division. Two observations worthy of comment occurred during remodeling of the redundant epithelium of the wound. A peculiar alteration in the appearance of approximately half of the cells of the basal layer was observed with phase and electron microscopes. Similar appearances were observed when wounds were fixed in osmic acid, glutaraldehyde or paraformaldehyde and when the osmolarities of these fixatives were varied. In addition, cells of this appearance were not present in the uninvolved basal layer of normal skin from the same block of tissue. Consequently, we believe the ultrastructural appearance is not due to an artifact of fixation, and represents a type of degeneration. Whether or not this type of degeneration is lethal to the cells remains to be seen, but its presence would certainly suggest either reduced metabolic activity or selective death of basal cells as a mechanism of remodeling redundant wound epithelium. Fallon and Saunders (1968) described selective cell death as a mechanism for remodeling of the digits during wing bud development in the chick embryo. Thus there is a precedent for this postulated mechanism for remodeling of wound epithelium. It is of interest that Ross (1970) has observed similar cells in his ultrastructural studies of wound healing. The second noteworthy observation was that although epithelial basement membrane antigen was absent during remodeling, an ultrastructurally unaltered basement membrane was present beneath the
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remodeled epithelium suggesting that a less extensive hydrolysis of the basement membrane was occurring. The inability to detect antigen cannot be due to lack of sensitivity of the immunohistochemical assay. Pierce et al. (1962) have demonstrated the presence of specific fluorescence within the cytoplasm of parietal yolk sac carcinoma cells in a pattern that correlated in distribution and amount with that of the endoplasmic reticulum. Thus very small amounts of antigen can be detected. Since degenerating epithelial cells of wounds (Grill0 and Gross, 1967) and of the gut of tadpole (Gross and Lapiere, 1962) hydrolyze collagen, it is probable that these degenerating cells also synthesize proteolytic enzymes that cause basement membrane to be partially hydrolyzed, and it is conceivable that antigenicity might be lost and definable ultrastructure remain. In support of this idea is the work of Michaeli et al. (1969), for example, which shows that antigenic determinants can be split from the collagen molecule without destroying the helical structure. SUMMARY
The healing of cutaneous wounds of mice was examined by immunofluorescent, phase, and electron microscopy. The results suggested that the epithelial basement membrane appeared to undergo degradation early in the healing process and did not regain all characteristics until remodeling of the redundant epidermis was completed. It did not appear to function as a substratum for epithelial migration; instead it appeared that the basement membrane may be partially responsible for the maintenance of the microenvironment of the epidermis. Phase micrographs illustrated a large number of degenerating basal cells at the completion of reepithelialization. These cells were confined to the basal layer and suggested that wound remodeling was initiated by selective cell death. These studies were supported by grants GM977 and AM13112 from Institutes of Health. The technical assistance of Mr. Marlin Nofziger, Jones and Mr. Howard Mitchell was deeply appreciated.
the National Mr. Alan S.
REFERENCES COONS, A. H. (1958). Fluorescent methods. In “General Cytochemical Methods” Danielli, ed.), pp. 399-435. Academic Press, New York. FALLON, J. F., and SAUNDERS, J. W., JR. (1968). In vitro analysis of the control death in the zone of prospective necrosis from the chick wing bud. Develop. 18, 533-570.
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GIACOMETTI, L., and PARAKKAL, P. F. (1969). Skin transplantation: Orientation of epithelial cells by the basement membrane. Nature (London) 223,514-515. GRILLO, H. C., and GROSS, J. (1967). Collagenolytic activity during wound repair. Deoelop. Biol. 15, 300-317. GROSS, J., and LAPIERE, C. M. (1962). Collagenolytic activity in amphibian tissues: A tissue culture assay. Boc. Nat. Acad. Sci. U. S. 48,1014-1022. KANG, A. H., NAGAI, Y., PIEZ, K. A., and GROSS, J. (1966). Studies on the structure of collagen utilizing a collagenolytic enzyme from tadpoles. Biochemistry 5, 509-515. KEFALIDES, N. A. (1969). Isolation and characterization of the collagen from glomerular basement membrane. Biochemistry 7, 3103-3112. MUKERJEE, H., SRI RAM, J., and PIERCE, G. B., JR. (1965). Basement membranes. V. Chemical composition of neoplastic basement membrane mucoprotein. Amer. J. Pathol. 46, 49-57. MICHAELI, D., MARTIN, G. R., KFITMAN, J., BENJAMIN, E., LEUNG, D. Y. K., and BLAT, B. A. (1969). Localization of antigenic determinants in the polypeptide chains of collagen. Science 166, 1522-1523. MIDGLEY, A. R., JR., and PIERCE, G. B., JR. (1963). Immunohistochemical analysis of basement membranes of the mouse. Amer. J. Fbthol. 43,929-943. ODLAND, G., and ROSS, R. (1966). Human wound repair. I. Epidermal regeneration. J.
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285-298. PIERCE, G. B., and NAKANE, P. K. (1969). Basement membranes. Synthesis and deposition in response to cellular injury. Lab. Znuest. 21,27-41. PIERCE, G. B., JR., MIDGLEY, A. R., JR., SRI RAM, J., and FELDMAN, J. D. (1962). Parietal yolk sac carcinoma: Clue to the histogenesis of Reichert’s membrane of the mouse embryo. Amer. J. Puthol. 41, 549-566. PIERCE, G. B., JR., BEALS, T. F., SRI RAM, J., and MIDGLEY, A. R., JR. (1964). Basement membranes. IV. Epithelial origin and immunologic cro8s reactions. Amer. J. Pathol. 45, 929-961. Ross, R. (1970). Personal communication. SAINTE-MARIE, G. (1962). A paraffin embedding technique for studies employing immunofluorescence. J. Histochem. Cytochem. 10, 250-256. SCH~LLING, J. A. (1968). Wound healing. Physiol. Rev. 48,374423. SCHMIDT, A. J. (1966). “Cellular Biology of Vertebrate Regeneration and Repair,” pp. 10-16. Univ. of Chicago Press, Chicago, Illinois. SPIRO, R. G. (1967). Studies on the renal glomerular basement membrane. Nature of the carbohydrate units and their attachment to the peptide portion. J. Biol. Chem. 242,1923-1932. SPIRO, R. G., and FUKUSHI, S. (1969). The lens capsule: Studies on the carbohydrate units. J. Biol. Chem. 244, 2049-2058. WESSELLS, N. K. (1963). Effects of extra-epithelial factors on the incorporation of thymidine by embryonic epidermis. Exp. Cell Res. 30,36-55. WINTER, G. D. (1964). Movement of epidermal cells over the wound surface. Aduan. Biol. Skin 5, 113-127.