REVIEW ARTICLE

AnatoW and Significance for Maintenance of Orderly

Tissue

Basal Lamina Scaffold:

Structure

Basal Lamina Scaffold-Anatomy and Significance for Maintenance of Orderly Tissue Structure
A Review
Rudolf Vracko, MD

The basal lamina is an extracellular scaffold positioned between parenchymal cells and connective tissue. Parenchymal cells attach to one of its surfaces and the other is anchored to connective tissue. By its presence it defines the spatial relationships among similar and disimilar types of cells and between these cells and the space occupied by connective and su ve tissues. Replenishment of cells which have died during normal functioning or have become damaged in course of injury occus with new cells in an orderly way along the framework of the basal lamina scaffold. This process appears to be aided by the polarity of the basal lamina and by an apparent specificity for cell types, and it enables multicellular organis to reconstitute histologic structures of most tissues and organs to what they were prior to loss of cells. If the basal lamina is destroyed, the healing in most tissues results in formation of scar and loss of function. The prerties of the basal lamIa cnrned with maintenance of histologic order in organs and tissues offer new ways to interpret the pathogenesis of several common disorders, including emphysema, scars, adhesions, cirrhosis of liver and excessive accumulation of basal lamina material as, for example, it occurs in patients with diabetes mellitus (Am J Pathol 77:313-346, 1974).

EACH TISSUE AND ORGAN of multicellular organisms is composed of several different types of cells and extracellular elements. These are arranged in precise relationships within the space of the organism and assume in each of the tissues and organs a characteristic histologic pattern. When cells die and drop out of this system, as the result of either cell senescence or injury, the organism has the ability to repair the ensuing defect. The process of repair may result in any of the following: a) complete reconstruction of anatomic relationships and reestablishment of function, where new cells are positioned in exact spatial relationships as they existed prior to injury; b) scarring and loss of function, where normal anatomic relationships of cells are not reestablished but the site of injury is populated by connective tissue or c) disorganized reconstruction, usually associated with impaired function, where new cells are positioned in a disorderly way, inadequately mimicking normal anatomic relationships.
From the Laboratory Service, Veterans Administration Hospital and the Department of Pathology, University of Washington School of Medicine, Seattle, Wash. Accepted for publication May 5, 1974. Address reprint requests to Dr. Rudolf Vracko, Veterans Administration Hospital, 4435 Beacon Ave South, Seattle, WA 98108.

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Recent experiments have shown that orderly positioning of new cells in different organs following cell death occurs by growth of cells along the scaffold of basal lamina.-1 It appears that if the basal lamina remains physically intact after loss of cells, it maintains by its presence the spatial plan of the tissue: it defines and contains the connective tissue space with one of its surfaces and with the opposite it provides a physical substrate for growth and orientation of proliferating parenchymal cells. If, on the other hand, the basal lamina is destroyed, orderly cellular reconstitution does not occur and the result is formation of scar and loss of function. Because of the importance the basal lamina seems to have for the maintenance of orderly tissue structure it is the purpose of this review to a) define the basal lamina and its anatomic distribution, b) describe its qualities concerned with maintenance of spatial relationships on the organisational level of cells and formed extracellular elements, c) cite experimental data which illustrate these properties and d) describe some of the disorders which seem to be related to defects in this system.
Definition of Basal Lamina

Ihe basal lamina (BL; synonyms: basement membrane, basement lamina) is generally defined by its anatomic location and structural appearance; as indicated in Text-figure 1, it is found wherever plasma membranes of parenchymal cells abut upon connective tissue. As far as we know, BL with this typical distribution is present in all multicellular organisms which evolve embryologically with three germ layers. For example, it is present in hydra, where it separates mesoglea from entoderm and ectoderm.4 As a sheet at the interface between parenchymal cells and connective tissue, one of the BL surfaces provides a substratum upon which parenchymal cells are positioned5' and differentiate,78 and the opposite surface is anchored to the connective tissue.5 Modified adaptations of this basic pattern are seen in renal glomemli, lung, liver and brain, and these will be described in detail. BL is applied intimately to the plasma membrane of parenchymal cells wherever their surfaces face connective tissue but does not extend between adjacent cells of same type. It is PAS-positive. With the electron microscope three zones can be distinguished:9 a) the lamina densa consists of tightly matted randomly oriented fibrils (3 to 4 mFt in diameter) embedded in a dense matrix; b) it is separated from parenchymal cells by a lighter zone (lamina rara or lucida), which is thought to contain elements responsible for attachment of BL to plasma membranes of

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TExT-FIG 1-Diagram of anatomic distribution of basal lamina (depicted as heavy black line) in its typical location between parenchymal cells and the space occupied by connective and supportive tissues. Parenchymal cells include all epithelial cells of epidermis and epidermal appendages, of the genitourinary, respiratory and gastrointestinal tracts, all exocrine glands, endothelial cells of the cardiovascular system, mesothelial cells of body cavities, cells comprising central and peripheral nervous systems, endocnne cells, muscle fibers and fat cells. The space occupied by connective and supportive tissue (shaded area) contains bone and associated cells, cartilage and associated cells, collagen, elastin and fibroblasts.

parenchymal cells;10 and c) on the opposite side of lamina densa is a zone characterized by larger fibrils, approximately 10 m[t in diameter, which appear to blend imperceptibly into collagen fibers of the adjacent connective tissue.9 The thickness of BL is uniform for a given structure, but differs among structures and animal species. It is resilient and tough and survives a variety of injuries."3,' When denuded of cells, it is a pliable sheet which crumples readily, forming folds and pleats. It also seems to possess some degree of elasticity-it appears to be thinner in distended capillaries than constricted ones.Y2 BL appears early in embryogenesis, forming as a product of molecular events at the inductive tissue interfaces."3"4 It appears to be primarily formed by parenchymal cells, although for in vitro formation of skin BL collagen must be present in the substrate.'5 Neoplastic cells on the other hand appear to have the capacity to synthesize BL in the absence of connective tissue."' Very little is known about the maintenance and intrinsic turnover of BL. Tritiated proline injected into normal rats is found within glomerular BL in 24 hours and remains there for between 3 to 6 months.17

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The chemical composition of BL is 90% protein, 8T carbohydrates and 2% lipids.18 There are approximately equal amounts of collagen-like and noncollagen proteins. The latter are glycoproteins containing glucose, galactose, mannose, fucose, hexosamine and some sialic acid. 17"920 There appear to be subtle quantitative and qualitative differences in chemical composition of BLs from different body sltes.19'20 It has been demonstrated that BL associated with epithelial cells contains histochemically and immunochemically identifiable substances which are produced by epithelial cells and probably represent surfaceassociated materials.21 Several types of antigens have been defined in the BL, including species-specific antigens for epithelial BL that do not cross-react, as well as common antigens that cross-react among species. The common antigens are associated with the pathogenesis of nephrotoxic nephritis.1'622
Basal Lamina Scaffold of Specific Organs In this section, the anatomy of BL scaffolds of skeletal muscle (Text-

figure 2), lung (Text-figure 3), kidney (Text-figure 4), parcreas (Textfigure 5), nervous system (Text-figure 6), skin (Text-figure 7) and liver (Text-figure 8) will be described, and experiments will be reviewed that deal with the functional significance of BL for those organs.
Skeletal Musce

Waldayer, in 1865 2 ascribed the role of guiding regenerating muscle cells to sarcolemmal tubes (definition of sarcolemma today: plasma membrane and BL of skeletal muscle fibers). In 1893, Volkman 24 showed that injured skeletal muscle will completely "regenerate" and become functional only if the "sarcolemma" remained intact; if it was damaged, the injury healed with a scar. Later, Clark25 demonstrated that "regeneration" of skeletal muscle fibers occurred in the direction of the old fibers, even if an excised piece of muscle was reimplanted at right angles to its original orientation. Vracko and Benditt 1 have recently reported on reconstruction of rat and rabbit skeletal muscles after injury with either freezing, ischemia or in sittu autografting. Each type of injury produces complete necrosis of cells. As depicted in Figure 1, BL remains intact in the area of injury and maintains a map of the outline of the spatial relationship between muscle fibers and capillaries. Repopulation of the defect with new cells occurs along the old BL (Figures 2 and 3), and the spatial relationships between cells as they existed prior to injury are reestablished 3 weeks

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TEmr-FG 2-Diam-am Of basal lamina scaffold of ietal muscle. Cross sections of three different sets of basal lamma tubes are seen: the largest tubes (M) contain skeletal muscle fibers; one set of the smaller ones (C) contain endothelial cell tubes of capilaries and the other (N) encompasses Schwann cells and axons of
hiS focaly divided intO tWO layers to form spaces which are no occupied by pericytes. Collagen in the connective tissue space (shaded area) attaches to the outside of all tUbes securing them in relative spatial posi-

PeriPhnal nerves. T1he caplary basal

tions.

after injury. This process appears to be aided by the ability of each category of cells to grow along the surface of its own BL. The new cells of muscle fibers and capillaries frequently form a new layer of BL which is of the usual thickness and is deposited primarily along the outer surfaces of plasma membranes in locations in which the new cells are separated from the old BL. Removal of old redundant BL layers appears to be mediated by interstitial fibroblast-like cells which apply themselves to the outer surfaces of skeletal muscle fibers." This mechanism of removal is more effective in skeletal muscle fibers than in skeletal muscle capillaries, with the result that skeletal muscle fibers generally have a single layer and capillaries have two layers 1 month after healing has been completed. Reconstruction of skeletal muscle occurs to some degree if excised portions are minced into 1 cu mm pieces and the fragments reimplanted into the defect. 2627 It is observed that in early stages of the repair process the newly proliferating immature muscle cells orient in the direction of implanted muscle fragments ' and that in later stages the muscle fibers are oriented in an irregular and disorderly'- fashion. These observations suggest that proliferating muscle cells are "threading" themselves through the short, randomly oriented fragments of BL tubes. The product is at best a partially reconstituted and nonfunctional skeletal muscle.27 To test whether the attraction of newly proliferating skeletal muscle

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cells for old skeletal muscle BL tubes is a cell-specific phenomenon or simply related to mechanical factors, we presented growing muscle cells with BL tubes belonging to other cell types. A portion of renal medulla in which cellular elements have been killed by freezing and thawing was implanted into a defect of skeletal muscle of the same animal. Renal medulla was chosen because its BL scaffold is similar to that of skeletal muscle consisting of parallel array of different caliber tubes: BL tubes of renal tubules are big enough to accommodate mature skeletal muscle fibers and those of interstitial capillaries fit in size capillaries of skeletal muscle. When the transplanted BL scaffold of renal medulla was examined 6 weeks after surgery it was found that skeletal muscle fibers did not grow into the empty BL tubes of renal tubules and ducts but instead the transplanted piece contained fibroblast-like cells and collagen (Figure 4). Capillaries from the skeletal muscle stump, however, did repopulate some of the preexisting BL scaffolds of vasa recta (Figure 5). It is of interest that these capillaries have the appearance of skeletal muscle capillaries, rather than renal medulla capillaries. If BL scaffold of skeletal muscle is destroyed by either severe crushing 25 28or cauterization with heat or strong acids 24 or if an excised piece of skeletal muscle is replaced by a fibrin clot,25 skeletal muscle fibers do not grow back but instead a scar forms in the area of injury. The regenerating muscle fibers from the viable muscle stumps grow in these experiments for about 1 mm in an irregular way and then stop. Scarring also occurs if injury to skeletal muscle is accompanied by infection. In our experiments, when the injured muscles became accidentally infected with bacteria the BL scaffolding was disrupted in many sites. Polymorphonuclear leukocytes were then present in large numbers and the injury eventually healed with a scar. It is not clear whether the presence of bacteria or white blood cells alone or a combination of the two caused the damage to the BL.
Lung

When cells die in the lung as a result of injury, BL provides a scaffold which guides the positioning of new cell generations. This was demonstrated by the following experiment:3 Cells in focal areas of dog lung involving approximately half the lung's parenchyma were killed by intravenous injections of oleic acid. The epithelial and endothelial BL remained intact following injury, maintaining among cellular debris a "map" of the lung's architecture as well as a structural continuity between living and dead portions of the lung (Figure 6). Three days after injury, reconstitution with epithelial and endothelial, and presumably

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TET-FIG 3-Diagram of basal lamina scaffold of pulmonary alveolar septi. The alveolar spaces (A) are defined by a continuous sheet of basal lamina which supports alveolar epithelial cells. The capillary basal lamina of interstitial capillaries (C) fuses with alveolar basal lamina to form a single layer of basal lamina. The interstitial connective tisse space (shaded area) contains collagen, elastin, interstitial cells and basal lamina tubes (N) of nerve fibers.

septal, cells began from the adjacent uninjured parenchyma, each cell type repopulated its "own" BL surface (Figure 7). Within 3 weeks most of the architecture and all measured functions of the lung were reestablished. The new parenchymal cells appeared to remove cell debris and unfolded the pleated BL in areas in which it was crumpled. Two types of lesions remained 3 weeks after injury: there were scattered foci of collagen deposition in which BL scaffolding still was present and undergoing cellular repopulations. There also were focal interruption of interalveolar septi giving rise to emphysematous enlargement of air spaces. The latter is of particular interest because interruption of BL, associated with an inability of epithelial cells to bridge the gap, provides a plausible explanation for pathogenesis of emphysema. Of further interest is the observation that, in the lung, in contrast to regenerating skeletal muscle or kidney, the repopulating cells did not deposit a new layer of BL but instead grew directly on the preexisting BL surfaces. There seem to be no experimental observations documenting the formation of scar after destruction of pulmonary BL scaffold. The observations that lung abscesses and caseating granulomas generally heal with formation of a permanent scar seem to support the notion that once the scaffold of the lung has been destroyed reconstruction does not occur.
K;ney

Kidney was probably the first organ in which the significance of BL for orderly cellular repopulation was recognized. Oliver2 pointed out that reepithelialization of renal tubules, in cases of acute tubular cell necrosis, occurs along the BL scaffolding from cells which have escaped injury. The repair was complete only if BL remained intact. If it was disrupted extensively, the tubules did not reconstitute into functioning

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TEx-FIG 4-Diagram of basal luaina scaffold of kidney. The endothelial basal lamina of afferent and efferent arterioles (A) join at the vascular pole of the glomemlus the basal lamina of Bowman's capsule to form a single layer of basal lamina of glomerular capillary loops (C). The basal lamina of Bowman's capsule continues at the urinary pole into the tubular basal lamina (T) which in turn continues into that of collecting ducts, renal pelvis, ureters, etc. The basal lamina pipes which form the scaffold of interstitial capillaries (I) are discontinuous, the openings corresponding to endothelial fenestrae. Not included in the drawing are spaces delineated by basal lamina which contain smooth muscle cells of arteries and arteriols.

units because regenerating tubular epithelial cells did not seem to have the ability to bridge large gaps in BL. When the gaps were small (size not specified) epithelial and mesenchymal tissues interacted to reform the wall of the tubule. MIany other examples of tubular reepithelialization following a variety of injuries have been described since then. Electronmicrographs from such studies frequently show that BL is "split" or 'layered" and cell debris is trapped between the layers.29 30 Figure 8 depicts the outcome of an experiment in which acute tubular cell necrosis was produced by freezing a portion of kidney in a rat which has received silver nitrate in its drinking water for 10 months. After injury the animal was given fresh water. The old BL, which was marked by silver deposits, became redundant, and new cells with unlabeled BL were seen to grow inside the old BL tube. In another series of experiments designed to determine if BL of glomeruli also provide a scaffold after cell death, portions of normal rat kidney cortex were frozen and thawed and were examined at intervals ranging from 24 hours to 6 weeks after injury. The acute lesion was characterized by destruction of most cells and preservation of BL scaffold of glomeruli (Figure 9). Preliminary results indicate that cellular repopulation began within 3 days, originating apparently from remaining cells in the glomeruli. Undifferentiated cells were seen in spaces de-

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fined by the endothelial surface of BL and equally undifferentiated cells filled Bowman's space. Cells in both compartments apparently removed cell debris and aligned themselves along the respective BL surfaces (Figure 10). Cells on the epithelial side of the BL at some point developed the characteristic features of podocytes and those on the capillary side became typical endothelial cells. Bowman's capsule also became repopulated by simple squamous epithelium. A new layer of BL was formed in apposition to the old one, sometimes in glomerular loops and frequently in Bowman's capsule.
Pancreas

Reconstitution of pancreas has been studied in detail in rats after iniury by either administration of ethionine while animals were kept on protein-free diet 31 or following resection of a portion of the organ.32 In rats, protein-free diet and intraperitoneal injections of ethionine for 10 days cause the death of many pancreatic acinar cells. Basal lamina, as well as the interacinar connective tissue, remain intact during and following the injurious event. Within 2 to 3 weeks acinar cells are replenished, and the normal gross and microscopic configuration of the organ is reestablished. In a parallel set of experiments, approximately 55% of rat pancreas body was surgically removed.32 Reconstitution of the restored portions did not occur, even in animals which were sacrificed 12 months after operation. There was an increase in weight of the residual piece of pancreas, suggesting that compensatory enlargement of the remaining pancreas had occurred. In another set of experiments,33 pancreatic acini became atrophic and
Tm-FiG 5-Diagram of basal lamina scaffold of pancreas. The acini (A) are lined by basal lamina sheet which is continuous with the basal lamina of ducts (D). The endocrine cells of islets of Langerhans (E) are surrounded by a basal lamina sheet wherever they are exposed to the connective tissue. It is not clear at present whether basal lamina of mature islets is in continuity with that of pancreatic ducts. Also shown are the typical basal lamina tubes of capillaries (C) and nerves (N). The collagen in the connective fissue space (shaded area) is anchored to the basal lamina surfaces.

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the ducts dilated 2 to 7 weeks after the main pancreatic duct was ligated. When the same pancreata were sampled 4 to 10 weeks after the main pancreatic duct was reopened, the gross and microscopic appearance of the organ had returned to normal. These experiments seem to indicate that cellular reconstruction of the pancreas will occur readily if the BL scaffolding is left intact following injury. If it is removed, as in surgical amputation, "regeneration" does not occur.
Nervous Systen

Axons of peripheral nerves will regrow after injury in an orderly rather than in random pattern along the tracts of old nerves, while axons in brain and spinal cord will not. In the central nervous system, axons sprout from proximal nerve stumps for a short distance and then stop, and the injury heals with scar formation.34, 35The suggestion has been

T=r-m 6-Diagram of basal lamina of nervous system. All surfaces of brain (B), spinal cord (S) and peripheral nerves (N) are lined by a continuous sheet of basal lanina. One surface of basal lamina is applied against ceIls of the nervous system and the other against collagen of surrounding connective tisse. When arteries (A) enter the brain they are surronded by leptomeningeal connective tissue. In capillaries (C), however, the basal lamina of capillary endothelium combmes with that of brain surface and obliterates the connective tissue space between them, and therefore only a single basal lamina layer is present between capillary endothelium and astrocytes. When capillaries become veins (V), the reverse is true: the comnwn basal lamina sheet separates, one leaf following the contours of brain surface and the other the contours of endothelium. The basal lamina of ventricles and that of ependymal lining (not depicted) is continuous with the basal lamina of brain surface.

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made that the reason for the different healing potential lies in the anatomic relationships: -" while peripheral nerve fibers, consisting of axons and Schwann cells, are encased in a BL tube which in turn is surrounded by collagen fibers (endoneurium), the nerve fibers in central nervous system, consisting of axons and oligodendrocytes, are in immediate apposition to other glial cells and their position within the space of the central nervous system is not defined by distinct BL tubes. When continuity of nerve fibers is disrupted, peripheral nerves and those in the central nervous system respond similarly in many respects. In both, degeneration of the axon in the distal and for a short distance in the proximal stump is followed by proliferation of encasing cells, ie, Schwann cells or oligodendrocytes.3"8 The critical difference between the two appears to be that Schwann cells proliferate along the path of the nerve in a tube defined by BL while glial cells proliferate only at the site of injury. When the axon which is sprouting from the proximal stump of a transsected peripheral nerve fiber finds the distal segment of the nerve, it grows rapidly, first on the surface between Schwann cells and BL and later encased by Schwann cells along the path defined by the BL, until the nerve fiber is reconstituted. The only visible residual of this process is frequently a new layer of BL inside the old BL tube.36'3 In brain and spinal cord, on the other hand, paths of distal nerve fiber segments seem to loose their identity after injury. Axons budding from proximal nerve stumps will grow there only for a very short distance within the area of injury, and the repair terminates as a rule by formation of a scar at the site of injury.
Sidn

The role of BL for the maintenance of cells and the repair of skin has not been studied systematically. It appears that cell renewal in normal skin occurs within the framework of its BL scaffold. For example, cyclic regrowth of hair bulbs and hair shafts takes place in the loci defined by the BL scaffolds of the involuted original hair shafts.40 When skin is injured, the damage may heal by scarring or by reestablishment of anatomic relationships as they existed prior to injury. Experiences with different types of skin injuries indicate that the depth of damage is probably the most significant factor which determines the outcome. For example if thermal injury is confined to cells of the epidermis (first-degree burn) and the basal cell layers and the BL are not damaged, the restoration of original conditions is rapid and complete. The presence of subepidermal bullae (second-degree burn) indicates that basal cells, the BL or both are damaged. The healing in such instances

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TExT-FIG 7-Basal lamina scaffold of skin. A continuous sheet of basal lamina separates epidermis (E) and skin appendages (S, H, SE) from the dermis. The contour of the boundary between epithehuim and the underlying connective tissue has the characteristic pattem of dermal papilla and rete ridges. The basal lamina dips into the dermis to provide scaffolds for several different types of skin appendages, each having distinct anatomic configuration. As in other organs, basal lamina is a substatum for the various types of parenchymal cells of the skin and skin appendages and is anchored with one of its surfaces to the connective tissue (shaded area). In the dermis, basal lamina scaffolds for capillaries (C) and nerves (N) are present.

may or may not result in complete restoration of original structures. And if epidermis, BL and subepidermal connective tissues are damaged, as it occurs in the third-degree burn, the healing is slow and a scar invariably forms. The fate of burns has been compared to healing of cutaneous wounds produced at skin graft donor sites.41 The speed and quality of the repair process bears a direct relationship to the graft's thickness: in sites from which thin split-thickness grafts have been removed, the healing is rapid with mild scarring, while in sites from which full thickness grafts have been taken, healing is prolonged, with severe scarring. The donor sites of thin split-thickness grafts retain most skin appendages and some tips of rete ridges. These can provide not only densely distributed foci from which new cells proliferate but also define by their presence substantial portions of the original boundry between dermis and epidermis. In pig skin, for example, from which a 0.2-mm-thick layer has been removed, the islands of skin appendages are separated maximally by 1.0 mm, thus breaking up the total wound surface area into a series of anastomosing microwounds.42 The other extreme, when most skin appendages are removed with the graft, the outgrowth of new epithelial cells will occur mainly from the grossly visible wound edges and spatial relationships between epidermis and dermis must be reestablished in a large area.

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It appears that whenever cells of the epidermis and the dermis establish a new interface the product is inferior: the rete ridges, dermal papillae and skin apppendages are not reformed; the epithelium is thinner; the dermal collagen is deposited in dense, irregularly placed bundles; and the characteristic subepidermal zone of connective tissue is absent. If such scarring involves a large contiguous area the grossly visible distortion of the skin surface is greater than if it involves small anastomosing segments of skin located between islands of normal skin. Cellular events on the recipient site of skin autografts and bomografts have been studied by Giacometti and ParakkaL43 They have shown that rhesus monkeys' epidermal cells grow from the adjacent viable epidermis of the host as well as from the hair follicles of the graft. In each case the newly formed cells migrate over the BL of the grafted epidermis, displacing the old epidermis, and attach to BL with hemidesmosomes. These authors feel that the substratum of transplanted BL provides "contact guidance," as postulated by Weiss,6 for orientation and positioning of epidermal cells. Another example of the apparent importance of BL for orderly repairing of skin structure is seen in healing of various types of bullous lesions." Blisters of pemphigus vulgaris and epidermolysis bullosa simplex, where BL remains intact and attached to the dermis, heal without formation of scar. In epidermolysis bullosa dystrophica (recessive type), where BL is part of the blister roof and therefore detached from the dermis, the healing results in scarring. In most lesions of erythema multiforme, the BL stays with the dermis, and the healing in general is without formation of a scar; however severe lesions in which BL may have been disturbed heal with the formation of a scar. The effect of the persistence of BL on the quality of repair and also its speed has been demonstrated by the following experiments: 45 intact suction-induced 5-mm large blisters of skin have an intact BL which becomes repopulated with several layers of epithelial cells within 24 hours following injury; in contrast, in open blisters the BL disappears as a structural unit and the base of the lesion becomes lined by cell debris and fibrin, and cell proliferation is markedly retarded. Although other factors such as drying and presence of cell debris must be considered as modifying factors, the absence of "contact guidance" 6 is prom ably the critical deficiencv.
Uver

Because BL is normally absent within the liver lobule, this organ represents a unique situation in the body. As was shown earlier, the BL

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Tm-'r-Fm S-iagam of basal lamina scaffold of normal liver. Epithelium of bile ducts and eium of liver vessels, induding those of arteries, arteriols, portal vein, and hepatic vein branches are all supported by basal lamina. In periportal areas where bile ducts jo liver ceUs and epithelium ons the lining of liver sinusoids, these basal lamina sheets terminate so that there is no basal lamina in the space of Disse. Whether this space is m open contiuity with the connective tissue space of portal areas as shown on the diagram or not is not clear. When injury occurs, a single layer of basal lamina is deposited in the space of Disse within hours after injury and seems to remain there until repair is completed.

scaffold in renal glomeruli, lung and brain capillaries have been modified by fusion of BLs belonging to two different cell types and by obliteration of the connective tissue space between them. In the liver the modification is apparently carried a step further: the BL between two different types of parenchymal cells, ie, liver cells and sinusoidal lining epithelium, is almost completely abolished, probably to facilitate exchanges between blood plasma and liver cells.-O Liver seems to possess, however, the capacity to form BL very quickly in response to injury. Alterman 47 observed with light microscopy that BL appears in the space of Disse of rat livers within 4 hours after ligation of bile ducts and 3 to 6 hours after partial hepatectomy. The BL remains present for 2 days and then diminishes, to disappear by the fourth day after injury. It is of interest that the presence of this temporary BL coincides in time with the maximal proliferation of liver cells in response to injury, such as occurs after partial hepatectomy.4748 In another set of experiments 49 in which guinea pig livers were injured by heat, electron microscopy revealed that BL was present in the space of Disse within 1 hour after injury. Later stages were not examined.

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In human livers altered by either viral hepatitis, fattv change or eirrhosis, Popper et al 50 found increased amounts of neutral and some acid mucopolysaccharides in the space of Disse which were interpreted as a matrix for deposition of collagen fibers. When similar liver lesions were examined with the electron microscope 46 increased amounts of collagen were present in the space of Disse, and the sinusoids were frequently "capillarized," ie, were composed of continuous layer of endothelium and a continuous investment of BL. So far no particular significance has been ascribed to the presence of the basal lamina which appears either following injury in experimental animals or in diseased livers of man. It is conceivable, however, that the presence or absence of BL in lobules of injured livers may determine whether the injurv will heal by reconstitution or by the formation of a scar. Possible implications of this idea will be discussed later.
The Fate of Injuries

Although the function of BL has not been carefully examined in all tissues and in many animal species, the evidence presented above seems to indicate that in many organs the outcome of healing events is determined by whether BL is present after injury or not. To be considered next are the differences among various types of healing processes, as thev occur in vertebrates, and the properties of BL which determine the final outcome of repair in injured organs and tissues. Reestablishment of normal anatomic and histologic relationships to what they were prior to injury occurs either by regeneration (ie, de novo formation of injured or amputated portions of organs) or by reconstitution (ie, repopulation of residual extracellular scaffold with new cells). The mode of repair depends on a) phylogenic status of the animal, b) type of organ or tissue and c) whether the acellular scaffold of the organ remains intact after injury or not. Some lower animals, including vertebrates of classes Amphibia and Reptilia, can grow back portions of complex organs which have been damaged or amputated, as for example, a newt's leg. This type of regeneration requires that amputated structures be replaced by outgrowth in situ of new parts which are equivalent to the original. The process generally begins with formation of blastema at the site of amputation and is followed by de novo formation of the lost portion. The process seems to closely recapitulate events which took place during embryo-

genesis.51'52 Higher vertebrates, including man, cannot reform or regenerate lost portions of complex organs, such as limbs, liver, pancreas, lung, kidnev

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or thyroid. The loss is generally compensated for bv hypertrophy and hvperplasia of the remaining uninjured portions. When, for example, three lobes of rat liver are amputated they do not regenerate but instead the remaining two lobes enlarge by compensatory hypertrophy and hyperplasia until the original liver cell mass is reestablished.48 The ability to regenerate is limited in higher vertebrates to certain portions of organs and tissues and includes two types of structures: those in which parenchymal cells, BL and connective tissue elements have to be formed anew and their spatial relationships reestablished, and a second category in which a new BL scaffold is not required because it is normally not present. In the first category the regeneration does not occur by dedifferentiation and blastema formation but by extension of preexisting structures. This is seen in endometrium which is reformed in every menstrual cycle by outgrowth from residual mucosa. The mucosa of the gastrointestinal tract also has the ability to grow and cover sites from which it has been lost, although the newly formed mucosa is frequently thinner and does not seem to possess the complexities of the original structure.-" Epidermis can also reform from preexisting cells; however, the product is thinner than normal and does not possess the usual pattern of rete ridges and lacks skin appendages. New capillaries will grow from preexisting ones53'54 and can transform into vessels with muscular walls.35 Smooth muscle cells of some organs also seem to have the ability to grow anew in an orderly way, as in ureters from which portions have been removed.55 Injuries to smooth muscle wall of bowel, on the other hand, heal with a scar.3' The cortex of adrenal gland (but not the medulla) can reform from a few adrenal cortical cells attached to the organ's capsule.35 Structures in which reformation of BL scaffold is not required for orderly reconstruction include bone, cartilage, tendons, lymphoid tissues and hematopoietic portions of the bone marrow. Since most organs of higher vertebrates will not "regenerate," ie, form de novo once portions have been lost, the fate of injury to these organs depends on whether the acellular scaffold of the organ has survived the injury or not. The repair proceeds accordingly and terminates in one of the following three outcomes: a) complete repair and reconstruction of damaged portions; b) formation of scar or c) disorganized proliferation of cells. The factors which seem to influence the outcome of these healing modes will be examined next.
Orderly Repair and Reconstruction

If cellular relationships are to be reestablished as they% existed prior to injury, the following three requirements must be met: a) the newly

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formed cells and extracellular elements must be positioned in exact spatial relationships as they existed prior to injury; b) the replacing cells must be of the same cell type as they were prior to injury; and c) the numbers of replacing cells must approximate closely the numbers present prior to injury. It appears that BL possesses properties necessary for meeting each of these requirements. It defines, by its presence, the relationships between spaces and surfaces occupied by parenchymal cells and the space occupied by connective tissue. It has polarity and specificity for cell types as indicated by experiments with injured lungs and skeletal muscle1 where the new cells grew exclusively on surfaces from which previous cell generations have shed. While it is possible that in some of these experiments the cells did not have access to the opposite side of BL, in experiments in which skeletal muscle was excised and reimplanted, the proliferating muscle fibers and capillaries had access to the opposite surface of BL but grew invariably inside and not on the outside of BL tubes. Furthermore, the new cells always appear to be of the type which was supported by that particular BL prior to injury. While it is conceivable that BL possesses the capacity to induce "uncommitted" cells to mature into cell types which are appropriate for that particular BL, it is more likely that cell-specific markers exist on the surface of BL which are recognized by cells and which identify the BL as belonging to a specific cell type.638 This is supported by the observation that proliferating skeletal muscle fibers do not recognize BL tubes belonging originally to cells of renal tubules and collecting ducts. It appears that BL does not only possess properties which determine the positioning of cells and cell specificity, but that it also may be concerned with induction and restriction of cell multiplication. When BL of skeletal muscle, lung and kidney becomes denuded of cells, the parenchymal cells begin to multiply and continue to do so as long as there is cell-free BL surface available. The proliferation stops as soon as the exposed surfaces are repopulated. The absence of cell-specific surfaces, devoid of cells, may then at least in part represent an important negative signal which sets the size limit to tissue reconstruction.
Formation of Scar

While presence of the BL facilitates reconstruction, its destruction generally causes the injury to heal by formation of a scar. In animal models in which skeletal muscle BL has been destroyed,24'2'28 the defect was replaced by scar tissue. Renal tubules will not heal by reconstitution following acute necrosis of tubular epithelial cells if the BL has

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been disrupted.2 Traumatic disruption of skin in which BL has not been carefully coapted will heal with formation of a scar, as will skin transplant donor sites from which BL has been removed; similarly burns which are deep enough to destroy BL and bullous lesions in which the BL is detached from the dermis also heal with scar formation. Reconstruction of normal anatomic relationships will also not occur if BL scaffold has been removed. Portions of pancreas which have been removed or destroyed will not be reconstructed, while complete reconstruction will occur if only parenchymal cells of the pancreas are killed, leaving the BL scaffold intact. Similarly, transection of nerve axons in spinal cord, where BL is absent, will result in a permanent scar,38 while reestablishment of normal anatomic relationships will occur in peripeheral nerve where BL is present. Scars or adhesions, as they occur, for example, in pleural and peritoneal cavities following infections or surgical procedures, could also be caused by breaks in the BL. It is known that defects in one peritoneal surface heal rapidly and completely; -w if, however, peritoneal surfaces are dried and a blood clot is introduced into the dried area, adhesions occur commonly.57 Although the fate of the BL in these experiments is not known, a reasonable possibility is that disruptions in the BL have occurred on opposing peritoneal surfaces through which connective tissue then establishes anastomosing bridges across the peritoneal space. A somewhat similar situations occurs in the kidney in the wake of glomerulonephritis, where cellular proliferations which obliterate Bowman's space as "crescents" are frequently accompanied by breaks in the BL of glomerular capillary loops.58 The breaks are presumably caused by neutrophilic leukocytes which are attracted to the glomerular BL by products of immunologic reactions.59 The source of cells which comprise "crescents" is unknown.
Disorganized Repair

The repair can result in disorderly and sometimes excessive proliferation resulting in distortion of normal anatomic structure. This occurs for example at the proximal stumps of amputated peripheral nerves where axons, Schwann cells and associated connective tissue proliferate in a disorganized pattem if the distal stump is absent. All these cells grow within a nodule (neuroma) where the new Schwann cells develop a new layer of BL. Although the relative positioning of the cells within the nodule may be accurate, the entire structure is curled up at the end of the nerve stump because the path of its growth has not been defined by a preexisting BL.

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Disorderly proliferation of parenchymal cells and of connective tissue as it occurs in cirrhosis of liver can be interpreted similarly. After injury of liver with either toxic or viral agents, the newly proliferating cells sometimes do not reestablish normal anatomic relationships but instead they became positioned in a disorderly way which only superficially mimicks nomal structure: liver cells grow in nodular aggregates rather than forming liver cell plates within the organizational structure of the liver lobule, and bile duct epithelium proliferates to form short, nonfunctioning bile duct branches. In addition, fibrous tissue is deposited in areas where liver cells have died. Observations that intact BL is an essential requirement for orderly cellular reconstitution of many injured organs and that liver cells are lined by BL only when injured, suggests the possibility that when cells of liver proliferate in disorganized patterns, something may have gone wrong with either formation or maintenance of the temporary BL scaffold. In the absence of a BL scaffold the replenishment of damaged liver cells would, according to this concept, not occur in structural harmony with uninjured portions of the liver, but instead liver cells would pile up in a disorderly way, distorting the normal anatomic relationships. Factors which may cause the absence of an effective BL scaffold in the injured liver could be inherent in the constitutional makeup of certain individuals and be expressed as failure to manufacture, preserve or recognize the temporary BL scaffold or they could be related to the duration and intensity of injury. The neutrophilic leukocvtes which appear in livers after prolonged and excessive consumption of alcohol in large numbers 60 could damage, as they do in acute glomerulonephritis,59 the BL scaffold.
Reduplication of Basal Lamina

The function of BL as a scaffold for orderly cell positioning appears to have an important role in pathogenesis of disorders associated with so-called thickening of BL. Almost invariablv, increased investments with BL are composed of multiple layers of BL rather than of a continuous and homogeneous widening of a single BL laYer. The term "thickenina," therefore, does not describe the process accuratelv. Observations made on capillaries from patients with diabetes mellitus 61.62 and experiments with skeletal muscle injury in nondiabetic animals 1 have revealed that excessive BL accumulation is a quantized phenomenon in which multiple, normally thick layers of BL are deposited in sequence

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by new cell generations. Further supporting this concept are observations on injured peripheral nerves,36'37 renal tubules (Figure 8), regenerating glomeruli 30,63 and other structures in which multiple layers of BL are present after the healing process has been completed. This phenomenon is not associated with every episode of cell renewal. Whether new cell generations will develop a new layer of BL or not appears to have some relationship to the distance which exists at a certain stage of cell development between plasma membrane of the newly formed cell and the old BL. If the plasma membrane is immediately apposed to the old BL, then a new BL layer is apparently not formed, but the old BL layer becomes the BL of the new celL If, however, the old BL is separated from the plasma membrane of the new cell by some distance, then the new cell develops a new layer of BL in at position to its plasma membrane. Wherever this occurs, the cell is lined by two or more layers of BL, as is depicted in Figures 3, 5, 8 and 11. At least two reasons for the nonalignment of new cells's surface with the old BL are apparent. In skeletal muscle fibers, for example, the old BL is thrown into folds and the plasma membrane of the new cell touches only the innermost surfaces of the BL folds and ridges. In regenerating capillaries of the skeletal muscle, on the other hand, the endothelial cell tube is separated from the BL of the original capillary by cell debris. Vhen the new layer of BL is formed, the cell debris may become encased between the two layers of BL.1 There also seem to be differences among tissues in regard to whether or not regenerating cells will produce a new layer of BL. Skeletal muscle fibers and muscle capillaries,1 renal tubules 29,30 and nerves 36'7 are examples of tissues where two layers of BL are commonly present after a single episode of injury and cellular reconstitution. In contrast, the alveolar epithelium and septal capillaries of the lung3 and the epidermis in the monkey 43 and mouse seem to utilize the old BL as their immediate footing and do not form a new layer of BL. The reasons for these differences are not apparent, and they do not seem to be related to the nature of injury. Another variable is that mechanisms exist for removal of the old BL once a new layer has been formed. This was observed in experiments with skeletal muscle injury, where several weeks after injury with freezing or ischemia, the muscle fibers had only one BL layer." This is not an instantaneous process, and the disappearance of old BL lags for some time after the removal of cell debris has been completed and cell reconstitution is well under way. It seems that the original BL of skeletal muscle fibers remains intact at least long enough to provide the template

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for harmonious reconstruction of the damaged muscle. The removal of the old BL appears to be mediated by fibroblast-like cells which become applied to the outer surfaces of newly developing muscle fibers.' The old BL is not invariably removed. In capillaries of skeletal muscle and in peripheral nerves, as well as in renal tubules, the outer layer of BL is still present weeks and months after injury, while in skeletal muscle fibers it is absent in samples examined approximately 3 weeks after injury. Although the reasons for the preferential removal of BL from skeletal muscle fibers has not been defined, an important stimulus may be that muscle cell contraction is not transmitted efficiently to tendons via collagen fibers attached to a loose and redundant BL. Should this notion be correct, then the primary function of the embracing interstitial cells is not the removal of old BL, but rather a remodeling of the attachments of collagen fibers from the old redundant to the new, tightly fitting BL. Excessive accumulation of BL occurs in several pathologic conditions of man. Of particular interest is the verv exaggerated BL accumulation in patients with diabetes mellitus. It occurs there in many structures which contain BL " and represents in capillaries the characteristic morphologic lesion of diabetic microangiopathy (Figure 11). Three explanations for this process seem possible: a) cells which ordinarily make only a single "normal" complement of BL may, under appropriate circumstances, have been "turned on" to produce new layers; b) cells which make ordinarily "normal" complement of BL only once in their life cycle have died and have been replenished by new cell generations which deposited a new layer of BL; or c) formation of BL is proceeding normally, but its removal is not functioning properly. The evidence, which has been presented in detail elsewhere,1'" -" 2strongly suggests that accelerated rate of cell death and cell replenishment accounts for most of accumulated layers. The underlying reason appears to be that cells of patients with diabetes mellitus die and are replenished at an accelerated rate, probably due to genetically transmitted cellular defect which renders diabetic cells excessively vulnerable to injurious events.' In many other situations in which cell death and cell replenishment occurs layering," "splitting" or "lamination" of BL in different organs and tissues appears to be a constant feature. In view of the evidence presented above, in most of these situations the individual layers of BL have probably been deposited there by new cell generations and give (like rings on cross sections of tree trunks) an indication of the number of cell generations which have occurred at that particular site. Whether there are other mechanisms responsible for quantized depo-

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sition of BL layers that are not associated with cell death and cell replenishment remains to be seen. It appears possible that short segments of new BL layers can be formed by cells, or portions of cells, which have become detached from their old BL support. The result would be, in such situations, a structure composed of anastomosing sheets of BL having a reticular appearance on cross section, similar to that of the aortic subendothelium in young rats.* There is no evidence at the present that excessive BL is deposited by uncontrolled and uninterrupted production of BL material; whether conditions exist in which either an aberrant chemical make up of BL prevents its resorption or, conversely, mechanisms for BL removal are not functioning properly is unknown. Questions of this nature, as well as others concerned with, for example, mechanisms responsible for maintenance of BL which has been denuded of cells or with the recognition systems which exist between cells and their BL, represent challenging problems for future studies.
References 1. Vracko R, Benditt EP: Basal lamina: the scaffold for orderly cell replacement. J Cell Biol 55:406-419, 1972 2. Oliver J: Correlations of structure and function and mechanisms of recovery in acute tubular necrosis. Am J Med 15:535-557, 1953 3. Vracko R: Significance of basal lamina for regeneration of injured lung. Virchows Arch (Pathol Anat) 355:264-274, 1972 4. Hess A, Cohen AL, Robson EA: Observations on the structure of hydra
as seen with the electron and light microscopes. Q J Microsc Sci 98:315-326, 1957 5. Pease DC: The basement membrane: substratum of histological order and complexity. Verhandlungen, Vierter Internazionale Kongress fiir Elektronmikroskopie, Berlin, Springer-Verlag 1960, pp 139-155 6. Weiss P: Guiding principles in cell locomotion and cell aggregation. Exp Cell Res (Suppl 8) :260-281, 1961 7. Gustafson T, Wolpert L: The cellular basis of morphogenesis in sea urchin development. Int Rev Cytol 15:139-214, 1963 8. Slavkin HC: The dynamics of extracellular and cel surface protein interactions, Cellular and Molecular Renewal in the Mammalian Body. Edited by IL Cameron, JD Thrasher. New York, Academic Press, Inc, 1971, pp 221-276 9. Majno G: Ultrastructure of the vascular membrane, Handbook of Physiology, Sec 2, Circulation, Vol III. Edited by W. F. Hamilton, P Dow. Baltimore, Williams and Wilkins, 1965 10. Tsao CH, Glagov S: Basal endothelial attachment: tenacity of cytoplasmic dense zones in the rabbit aorta. Lab Invest 23:510-516, 1970 11. Ross MH, Grant L: On the structural integrity of basement membrane. Exp Cell Res 50:277-285, 1968 12. Vracko R: Skeletal muscle capillaries in nondiabetics: a quantitative analysis. Circulation 41:285-297, 1970

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13. Grobstein C: Mechanisms of organogenetic tissue interactions. Natl Cancer Inst MIonogr 26:279-299, 1967 14. Kallman F, Grobstein C: Localization of glucosamine incorporating materials at epithelial surfaces during salivary gland epithelio-mesenchvmal interaction in vitro. Dev Biol 14:52-67, 1966 15. Briggaman RA, Dolldorf FG, Wheeler CE Jr: Formation and origin of basal lamina and anchoring fibrils in adult human skin. J Cell Biol 51:384-395, 1971 16. Pierce GB, Nakane PK: Antigens of epithelial basement membranes of mouse, rat and man: a study utilizing enzyme-labeled antibody. Lab Invest 17:499-514, 1967 17. Lazarow A, Speidel E: The chemical composition of the glomerular basement membrane and its relationship to the production of diabetic complications, Small Blood Vessel Involvement in Diabetes fMellitus. Edited by MD Siperstein, AR Colwell, K Meyer. Washington, DC, American Institute of Biological Sciences, 1964 18. Berman LB, Misra RP: NMolecular nephrology, Editorial. Am J Med 53: 701-703, 1972 19. Kefalides NA: The chemistry and structure of basement membranes. Arthritis Rheum 12:427-443, 1969 20. Kefalides NA, Winzler RJ: The chemistry of glomerular basement membrane and its relation to collagen. Biochemistry 5:702,713, 1966 21. Bernfield MR, Banerjee SD, Cohn RH: Dependence of salivary epithelia] morphology and branching morphogenesis upon acid mucopolysaccharideprotein (proteoglycan) at the epithelial surface. J Cell Biol 52:674-689, 1972 22. Krakower CA, Greenspon SA: The antigens of capillary and vascular basement membranes elucidated by the use of lens capsule."7 23. Waldeyer WV: Vber die Veranderungen der quergestreiften Muskeln bei der Entziindung und dem Typhus-prozess, sowie fiber die Regeneration derselben nach Substanz-defekten. Virchows Arch Pathol Anat Physiol Clin Med 34:473, 1865 24. Volkman R: tJber die Regeneration des quergestreiften 'Muskelgewebes beim Menschen und Saugethier. Beitr Pathol Anat Allg Pathol 12:233, 1893 25. Clark WE, Le Gros: Experimental study of regeneration of mammalian striped muscle. J Anat 80:24-36, 1946 26. Carlson BM: Regeneration of completely excised gastrocnemius muscle in the frog and rat from minced muscle fragments. J Morphol 125:447-472, 1968 27. Kaspar U, Mumenthaler M, Steiner A, Ludin HP, Wiesmann U: Regeneration of tibialis anterior muscle in rat after complete excision and reimplantation of muscle fragments. Z Neurol 200:18-32, 1971 28. Allbrook D: An electron microscopic study of regenerating skeletal muscle. J Anat 96:137-152, 1962 29. Cuppage FE, Neagoy DR, Tate A: Repair of the nephron following temporary occlusion of the renal pedicle. Lab Invest 17:660-674, 1967 30. M.adrazo A, Suzuki Y, Churg J: Radiation nephritis. II. Radiation changes after high doses of radiation. Am J Pathol 61:37-56, 1970 31. Fitzgerald PJ, Herman L, Carol B, Roque A, 'Marsh WH, Rosenstock L, Richards C, Perl D: Pancreatic acinar cell regeneration. I. Cytologic, cvtochemical and pancreatic weight changes. Am J Pathol 52:983-1011, 1968 32. Lehv M, Fitzgerald PJ: Pancreatic acinar cell regeneration. IV. Regeneration after surgical resection. Am J Pathol 53:,513-535, 1968

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305-325, 1963 54. Schoefl GI, MIajno G: Regeneration of blood vessels in wound heahing.4- pp 173-193 55. Kiviat MD, Ross R, Ansell JS: Smooth muscle regeneration in the urether: electron microscopic and autoradiographic observations. Am J Pathol 72:403416, 1973

33. Tiscomia OM, Jacobson JH, Dreiling DA: Microsurgery of the canine pancreatic duct: experimental study and review of previous approaches to the management of pancreatic duct pathology. Surgery 58:58-72, 1965 34. Ramon y Cajal S: Degeneration and Regeneration of the Nervous System. New York, Hafner Publishing Co, 1959 35. McMinn RMH: Tissue Repair. New York, Academic Press, Inc, 1969 36. Thomas PK: Changes in the endoneurial sheaths of peripheral myelinated nerve fibers during Wallerian degeneration. J Anat 98:175-182, 1964 37. O'Daly JA, Imaeda T: Electron microscopic study of Wallerian degeneration in cutaneous nerves caused by mechanical injury. Lab Invest 17:744-766, 1967 38. Lampert P, Cressman M: Axonal regeneration in the dorsal columns of the spinal cord of adult rats. Lab Invest 13:825-839, 1964 39. Parakkal PF: The fine structure of anagen hair follicle of the mouse. Advances in Biology of Skin, Vol 9, Hair Growth. Edited by W Montagna, RL Dobson. New York. Pergamon Press, Inc, 1969, pp 441-469 40. Parakkal PF: Ultrastructural changes of the basal lamina during the hair growth cycle. J Cell Biol 40:561-564, 1969 41. Converse JMI, Robb-Smith AHT: The bealing of surface cutaneous wounds: its analogy with the healing of superficial bums. Ann Surg 120:873-885, 1944 42. Winter GD: NMovement of epidermal cells over the wound surface. Advances in Biology of Skin, Vol 5, Wound Healing. Edited by W Montagna, RE Billingham. New York, Pergamon Press, 1964, pp 113-127 43. Giacometti L, Parakkal PF: Skin transplantation: orientation of epithelial cells by the basement membrane. Nature 223:514-515, 1969 44. Lever WF: Histopathology of the Skin. Philadelphia, JB Lippincott Co, 1961 45. Krawczyk WS: A pattern of epidermal cell migration during wound healing. J Cell Biol 49:247-263, 1971 46. Schaffner F, Popper H: Capillarization of hepatic sinusoids in man. Gastroenterologv 44:239-242 1963 47. Alterman K: Some local factors in the restoration of the rat liver after partial hepatectomy. AMA Arch Pathol 53:197-208, 1952 48. Bucher NLR: Regeneration of mammalian liver. Int Rev Cytol 15:245-30, 1963 49. Cossel L: tYber akutes Auftreten von Basalmembranen an den Lebersinusoiden. Beitr Pathol 134:103-122, 1966 50. Popper H, Paronetto F, Barka T: PAS-positive structures of nonglycogenic character in normal and abnormal liver. AMA Arch Pathol 70:300-313, 1960 51. Schmidt AJ: Cellular Biology of Vertebrate Regeneration and Repair. Chicago, University of Chicago Press, 1968 52. Goss RJ: Principles of Regeneration. New York, Academic Press, 1969 53. Cliff WJ: Observations on healing tissue: a combined light and electron microscopic investigation. Philos Trans R Soc Lond (Biol Sci) Series B 246:

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56. Eskeland G, Kjaerheim A: Regeneration of parietal peritoneuim in rats. II. An electron microscopial study. Acta Pathol Microbiol Scand 68:379-395, 1966 57. Ryan GB, Grobety J, Majno G: Postoperative peritoneal adhesions: a study of the mechanism. Am J Pathol 65:117-148, 1971 58. Mortia T, Suzuki Y, Churg J: Structure and development of the glomerular crescent. Am J Pathol 72:349-368, 1973 59. Cochrane CG: Immunologic tissue injury mediated by neutrophilic leukocytes. Adv Immunol 9:97-162, 1968 60. Rubin E, Lieber CS: Fatty liver, alcoholic hepatitis and cirrhosis produced by alohol in primates. N Engl J Med 290:128-135. 1974 61. Vracko R, Benditt EP: Capillary basal lamina thickening: its relationship to endothelial cell death and replacement. J Cell Biol 47:281-285, 1970 62. Vracko R: Skeletal muscle capillaries in diabetics: a quantitative analysis. Circulation 41:271-283, 1970 63. Porter KA, Dossetor JB, Marchioro TL, Peart WS, Rendall JM, Starzl PI: Human renal transplants. I. Glomeruar changes. Lab Invest 16:153-181, 1967 64. Vracko R: Basal lamina layering in diabetes mellitus: evidence for accelerated rate of cell death and cell regeneration. Diabetes 23:94-104, 1974 65. Vracko R, Benditt EP: Manifestations of diabetes mellitus: their possible relationships to an underlying cell defect. Am J Pathol 75:204-223, 1974 66. Schwartz SM, Benditt EP: Postnatal development of the aortic subendothelium in rats. Lab Invest 26:778-786, 1972

Acknoledgments
Excelent technical assistance has been provided by Ms. Alice Yasutake and Ms.

Marli Knox.

Legends for Figures

Fig 1-Electron micrograph of rat gracilis muscle cross-section 4 days after a portion of it was frozen and thawed. Section is from central area of injury where cellular response to injury is still absent All original cells have disintegrated. Cell debris of muscle fibers (M) and capillaries (C) is seen within an intact, and sometimes pleated, basal lamina. The debris is in various stages of degradation and is absent (artifact?) from some basal lamina tubes. An apparently intact red blood cell is in one capillary (left upper corner) and several red blood cell ghosts (R) are seen among collagen fibers of the interstitial connective tissue (x 4900).
Inside the pleated basal lamina tube are several cell bodies, each containing an abundance of fibrils. At a later stage of development not shown here (for details see Vracko and Benditt1) these cells fuse to form a single cell body which occupies the cross-section of the newly formed muscle fiber. The microfilaments increase in density to give the cell the characteristic morphologic feature of skeletal muscle fiber. A new layer of basal lamina is formed at this stage in sites where the old pleated basal lamina is some distance away from the plasma membrane of the new cell (x 5700).

Fig 2-Rat gracilis muscle fiber undergoing repair 1 week following injury with cold.

Fig 3-Cross-section of a capillary from gracilis muscle of rat 5 weeks after a piece of muscle was excised and reimplanted. The 20-month-old animal was given .15 M silver nitrate solution as drinking water from weaning until the day of operation and fresh water after operation. The resulting accumulation of silver granules (open arrows) is within the old, now outer, layer of basal lamina indicating that the inner silver-free layer (solid arrows) formed after the operation. Similar reduplication of BL is also apparent in two of the three muscle cells (x 11,200).

Fig 4-Electronmicrograph of rabbit kidney medulla 3 weeks after it has been removed, frozen and thawed and implanted into a defect made in the anterior tibial muscle of the same animal. Muscle cell from the uninjured portions of the muscle did not grow into the basal lamina tubes of collecting ducts and loops of Henley (T). Instead, fine fibrils, presumably collagen, were deposited inside and outside of the tubes by fibroblast-like cells. The layering of basal lamina in some of the tubules probably was present prior to freezing injury (x 3600).

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muscle into the interstititum of renal medulla. Such capillaries resemble those of skeletal muscle since they have a continuous layer of endothelium and a continuous layer of basal lamina (solid arrows). The entire structure is in tum surrounded by discontinuous fragments of basal lamina (open arrows) which are similar in appearance to basal lamina of interstitial capillaries of renal medulla. Finely fibrillar material and fibroblast-like cells (F) are present throughout the area occupying the inside and outside the basal lamina of renal tubules (T) (x 7100). Fig 6-Dog lung 24 hours after the animal received intravenous injection of oleic acid. All cells except red blood cells have disintegrated, leaving behind intact basal lamina scaffold which preserves the spatial relationships among different structures. The capillary basal lamina (solid arrows) contains cell debris of capillaries (C) and the alveolar basal lamina (open arrows) defines with one of its surfaces the interstitiai compartment (S) and with the other, the alveolar spaces (A). The two basal laminas are frequently fused to form a single layer (X) (x 7400) Fig 7-Dog lung 5 days after it was injured with intravenous injection of oleic acid. The basal lamina scaffold is intact and seems to provide guidance to proliferating cells. Inside the capillary basal lamina tubes (solid arrows) are poorly differentiated cells with prominent endoplasmic reticulum and phagolysosomes. Most cell debris inside of capillary spaces (C) has been removed and lumens have not yet formed. In the alveoli (A) epithelial cells (E), some of which have villi, are covering part of alveolar basal lamina, while some of basal lamina surfaces are still denuded (open arrows). In the septal interstitium (S) collagen and elastin are present as well as a portion of an undifferentiated cell and an extravasated red blood cell. Three weeks after injury most of the basal lamina scaffold in this experimental model is repopulated with appropriate cell types and the anatomic structure and function of the lung are reestablished (x 5800).

Fig 5-From the same tissues as Figure 4 showing a capillary (C) growing from uninjured

Fig 8-Portions of two adjacent proximal convoluted tubules from a rat 3 weeks after part of kidney cortex was frozen and thawed. The 8-month-old rat received .15 M silver nitrate

solution as drinking water from weaning until the day of injury and fresh water after operation. The resulting accumulation of silver grains is in the outer, redundant basal lamina (open arrow) while the cells of the tubules are lined by a delicate, unlabeled basal lamina (solid arrows) which presumably has been formed after injury. Between the old and new layers are present cell debris from the original cell population, residual portions of cells and Fig 9-Portion of collagen fibers. The source of collagen fibers is unknown (x 10,000). rat glomerulus 24 hours after part of renal cortex has been frozen and thawed. Most cells in the area of injury have disintegrated leaving behind cell debris and basal lamina scaffolds of renal tubules (T), Bowman's capsule (arrow) and glomerular capillary loops (C) (x 2600). Fig 10-Portion of rat glomerulus from an area of renal cortex that has been frozen and thawed 2 weeks previously. The original basal lamina scaffold is still intact The spaces of capillaries (C) and Bowman's space (B) are being repopulated with new cells that seem to become aligned along the surfaces of basal lamina. New cells also repopulate renal interstitium and the basal lamina scaffold of renal tubules (T). In latter stages of glomerular repair, the newly formed cells seem to differentiate into typical endothelial cells, podocytes and Bowman's epithelium, and the only remaining evidence that injury has taken place is focal reduplication and focal puckering of basal lamina. In some portions of injured cortex, extensive calcification of cell debris seem to prevent cellular reconstitution in spite of the presence of structurally intact basal lamina scaffold (x 4500).

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Fig 11 A cross-section of capillary from anterior tibial muscle of a 70-year-old diabetic who was treated with insulin for 15 years. Specimen was obtained 12 hours postmortem. The markedly increased investment with basal lamina is caused by apposition of multiple, concentric layers of basal lamina separated by spaces which contain either cell debris (arrows), loosely textured fibrillar material or collagen fibers (lower portion of the capillary circumference). The source of collagen is unknown. Some of the cell debris containing spaces between basal lamina layers have the configuration of pericytes. It is apparent that in some locations of the circumference the layers of basal lamina are fused, giving the appearance of homogeneous thickening. The innermost layer is applied intimately to the outer surfaces of endothelial cells (E) and pericytes (P) and represents the chronologically youngest deposition (x 16,000).