Report
ULTRASTRUCTURE OF KELOID: AN UNUSUAL INCIDENT INVOLVING LEPROMATOUS LEPROSY ROBERT F, DYER, Ph,D,, AND CARL D, ENNA, M,D, From the Department of Anatomy, Louisiana State University Medical Center, New Orleans, and the Department of Surgery, USPHS Hospital, CarviUe, Louisiana
ABSTRACT: A patient w/fh lepromatous leprosy developed keloids on the dorsum of both arms in response to ulcerations due to acute erythema nodosum leprosum reactions. Electron m/croscop/c exam/nation of the keloidal dermis showed a morphology indicative of increased production of normal collagen fibrils. The greatest cellular changes from normal were in fibroblasts which were enlarged due to increased amounts of rough endoplasmic reticulum and extensive Colgi complexes. Nuclear folds were also evident in these fibroblasts. Some cells, considered to be fibroblasts, were filled with cytoplasmic fifamehts and contained bizarre shaped nuclei. Mast cells, blood vessels and nerve processes were also present.
lepromatous leprosy usually form scars that are flat, atrophic and nonadherent, apparently due to the disruption and destruction of connective tissue elements by the disease infiltrate. However, a patient at the USPHS Hospital at Carville, Louisiana, did manifest keloids on the posterior aspect of the upper arms in response to ulcerations caused by acute erythema nodosum leprosum reactions. Since biopsy specimens were necessary for diagnosis, we had the opportunity to investigate the ultrastructure of a condition that rarely occurs in leprosy patients. In this article we will discuss tine ultrastructural features of keloid in general, and specifically in relation to this leprosy patient.
Keloids and hypertrophic scars develop in traumatized human skin when the normal equilibrium between collagen synthesis and degradation is disrupted. Biochemical studies have shown that this disruption favors excessive collagen synthesis by fibrobiasts of the involved derm i s . i Ultrastructural studies of hypertrophic scars support the biochemical evidence, because the majority of fibroblasts depict a morphology indicative of h i g h rates of protein synthesis.^ A similar ultrastructurai morphology probably exists for keloids, but electron microscopic studies of this type of scar are lacking.
Materials and Methods The patient, a 21-year-old Caucasian man with active lepromatous leprosy, was admitted to the USPHS Hospital at Carville. Ulcerations due to acute reactions developed on the dorsal aspect of both arms, and the healing process resulted in excessive scar formation that continued to enlarge with time. Biopsies
Keloids rarely form in patients with lepromatous leprosy, even though the denervated skin of these patients is exposed to frequent injury. Patients with
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of the scars were removed approximately 18 months after the initial ulcerations, and the diagnosis of keloid was confirmed after gross and light microscopic examination. Portions of the scar tissues were also prepared for electron microscopy. Fresh samples were placed in cold 4% glutaraldehyde buffered to pH 7.2 with 0.2 m phosphate buffer, fixed for at least 24 hours, and rinsed in 0.2 m phosphate buffer containing 0.3 m sucrose. The dermis and epidermis were separated, and the dermal tissue was diced into 1 mm cubes and post-fixed for 2 hours in 1% osmium tetroxide buffered to pH 7.2 with 0.2 m phosphate buffer. The samples were dehydrated in a graded series of alcohols, equilibrated with propylene oxide, and embedded in Spurr embedding medium. Thin sections were cut on a Reichert OMU2 ultramicrotome, stained with lead and uranyl ions, and viewed with a Philips EM 200 electron microscope. Results
The morphology described is restricted to the reticular layer of the keloidal dermis. A sample of nonkeloidal dermis from an adjacent site did not reveal any of the unusual ultrastructural features characteristic of the keloid. Therefore, it is assumed that the results reflect dermal changes related to keloid formation rather than to lepromatous leprosy. Figure 1 shows that the structure and organization of connective tissue cells and fibers were disrupted in the keloidal dermis. Fibroblasts were enlarged, and collagen closely invested all cellular elements, obliterating spaces normally seen between collagen bundles and around cells. Collagen fibrils were usually ori-
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ented in the same direction, but deviations from this parallel arrangement occurred, especially adjacent to connective tissue cells (Figs.^2, 5, 8). The fibrils approximated 1000 A in diameter, had a normal periodicity of 640 A, and interfibriilar matrix material was scant. Elastic fibers were not demonstrated in the keloidal dermis, a condition that is characteristic of keloids in general. Three categories of connective tissue cells were obvious in the keloidal dermis: (1), cells comprising the fibroblast population, (2), cells of the macrophage system, and (3), the mast cell population, usually associated with neurovascular structures. The fibrobiast population was composed of a number of cell types that are considered to reflect different functional states of the same cell. One type had the structure of fibrocytes. These cells were flattened, had an attenuated cytoplasm with few organelles, and a smoothsurfaced nucleus that conformed to the cell outline. The nucleus sometimes contained a well-developed nucleolus (Fig. 2). Another cell type in the first category was identifiable as very active fibroblasts. These cells were the predominant cell population in the keloid dermis, and they were enlarged by accumulations of rough endoplasmic reticulum and Colgi complexes (Fig. 3). Nuclear volume also appeared increased, although changes in cell shape could have accounted for this impression in a single plane of section. More obvious changes were projections and indentations of the nuclear envelope (Fig. 3), in contrast to the smooth-surfaced nuclei of fibrocytes (Fig. 2). Higher magnification of the active fibroblast cytoplasm showed large Golgi
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Fig. 1. Vievi' of reticular layer of keloidal dermis showing a fibroblast (F) and portions f other cells embedded in a dense collagen matrix (C) ( x 5000).
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Fig. 2. Top, a fibrocyle exhibiting a smooth surfaced nucleus (N) containing a prominent nucleolus (Nu). Collagen (C) is seen in different planes of orientation (X 2600). Fig. 3. Bottom, a fibroblast enlarged by numerous Golgi complexes (G) and cisternae of rough endoplasmic reticulum (ER). The nucleus (N) has an irregular surface (X 7500).
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Fig. 4. Top, high magnificalion of fibroblasi (yU)|)l,isni showing a Golgi zone (C) composed of vacuoles (V) and smaller vessels (VE), Microfilaments (MF) and microtubules (arrows) are seen, (ER, rough endoplasmic reticulum) (x 12,500), Fig, 5, Bottom, fibroblast stained with phosphotungstic acid. Collagen is seen extracellulary in different planes of section (C) and also the cytoplasm of the fibroblast (C with arrows) (X 12,500).
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complexes consisting of numerous vacuoles and vesicles (Fig. 4). Tbe larger vacuoles had no visible content, some medium-size vacuoles contained a flocculent precipitate, and the smaller vesicles were a heterogeneous population that included coated vesicles. The profiles of rougb endoplasmic reticulum, although numerous, were generally not dilated, and the cisternae contained a precipitate similar to tbat of the Colgi vacuoles (Fig. 4). The cytoplasm of tbese cells also had variable amounts of microfilaments and a few microtubules (Figs. 4, 5). In addition, fibroblasts sometimes contained collagen fibrils witbin their cytoplasm (Fig. 5), a feature of fibroblasts involved in collagen remodelling associated with wound repair.^ Certain cells witbin the keloid had an unusual morphology, but were still considered components of the fibroblast population. They contained pleomorpbic nuclei and large amounts of rougb endoplasmic reticulum, but their predominant cytoplasmic feature was an abundance of randomly organized microfilaments (Fig. 6). Otber cells of this type contained a larger population of microfilaments that were more organized in appearance (Fig. 7). Tbese cells contained fewer cytoplasmic organelles, but distention of rough endoplasmic reticulum was a dominant morphological feature. In other examples of this peculiar cell type (Fig. 8), the nucleus was large and pleomorphic, with a well-developed nucleolus. The cytoplasm was scant relative to other fibroblast cells and mostly occupied by a feltwork of microfilaments. Tbe organization of the microfilaments and the presence of small densities along their length was similar to that seen in myofibroblasts of contracting granulation tissue;"* however, other features of tbe myofibroblast are not
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evident, and tbese cells were considered a part of the fibroblast population. The general category of cells, wbich were components of tbe macrophage system, were considerably fewer than the active fibroblasts. The cytoplasm of the macrophages contains scattered profiles of rough endoplasmic reticulum and some microfilaments. Lysosomes and phagocytic vacuoles were only occasionally present in tbe peripheral cytoplasm, but an obvious cytoplasmic feature of many macrophages was a vacuolization in tbe region of tbe nucleus (Figs. 9, 10). These vacuoles varied in size and content; some appeared clear, others contained a homogeneous material, and others contained membranous whorls (Fig. 10). Occasionally the nuclei of the macrophages contained nuclear inclusions (Fig. 9). Tbe third category of cells, the mast cells, occurred in large numbers in association with blood vessels and accompanying nerves tbat coursed through the keloidal dermis (Fig. 11). The cells contained granules that exhibited a homogeneous internal structure, or a crystalline or scroll-like membrane pattern. Degranulated mast cells were rarely seen in the keloidal dermis. Small blood vessels and related pericytes were common in keloidal dermis (Fig. 11), but the lumina were often collapsed and red cells were infrequent. Blood vessels were anticipated in the tissue, but nerve processes were not; however, in this case, small intact nerve bundles were usually found in the company of mast cells and blood vessels. Usually tbese nerve bundles consisted of unmyelineated axons with an associated Schwann cell (Fig. 11). These bundles were frequently embedded in a collagen fibril matrix, which in turn was invested by a connective tissue cell (Fig. 11).
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Fig. 6. Type of fibroblast containing an irregular nucleus (N) and many unorganized microfilaments (MF) (X 26,600).
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Fig. 7. Top, type of fibroblast exhibiting a pleomorphic nucleus (N) and distended cisternae of rough endoplasmic reticulum (ER), Bundles of microfilaments (MF) occupy the cytoplasm ( x 19,200), Fig, 8, Bottom, fibroblastic cell with some characteristics of a myofibroblast. Small densities which are similar to contraction bands are seen at the arrows. (N, nucleus; Nu, nucleolus; C, collagen) ( x 7500).
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Fig. 9. Top, cell identified as a macrophage. Accumulations of cytoplasmic vacuoles (CV) are seen in close relationship to the nucleus (N), (NI, nuclear inclusions) (X 4500), Fig. 10. Bottom, macrophage exhibiting cytoplasmic vacuoles (CV) surrounded by the nucleus (N). The fibrous lamina of the nucleus (FL) and the associated outer leaflet of the nuclear envelope completely surround the vacuoles (X 9900).
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Fig. 11. Top, mast cells (MC) in company with a small blood vessel and nerve, Tbe lumen of the vessel (L) is collapsed and pericytes (P) are seen, Tbe nerve bundle (NB) is surrounded by collagen (C) and a connective tissue cell (CTC), (E, endotbelial cell) ( x 4500), Fig. 12. Bottom, a single axon (AX) enveloped by its Scbwann cell (SC) ( x 17,000).
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Other nerve processes were found at a distance from the vascular network, but these were usually single axons that contained mitochondria and many neurofilaments. These axons were frequently enveloped by a Schwann cell (Fig. 12). Discussion The gross and the histologic appearance of keloids are well documented,'' b u t there is controversy over the classification of this type of excessive scar. However, most investigators consider keloids and hypertrophic scars to be manifestations of the same process.2' ^ Hypertrophic scars have been studied a n d characterized with the electron microscope,-' ""' but to our knowledge similar studies on keloids, particularly in patients with leprosy, are not available. Although our study is limited to mult i p l e lesions from a single patient, it is possible to state that the electron microscopic features of these keloid lesions d o not differ significantly from those of hypertrophic scars. It may be of greater importance to contrast the structure of keloids and hypertrophic scars to normal scars. The gross differences are obvious, and the histologic features of excessive scars and normal scars permit distinctions to be made, but neither of these levels of observation provide a clear insight into factors responsible for excessive scar formation. Biochemical and ultrastruct u r a l studies indicate that excessive scars are a result of increased collagen production by hypertrophied fibroblasts that have probably also undergone hyperplasia. This fibroblastic response is not unique to keloids and hypertrophic scars, since a similar response occurs in normal scar formation.'" However, the response is maintained for abnormal periods of time in those instances in which excessive scars are formed. The fibroblasts produce the fibrous elements that
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result in massive scar formation, but it may be that this reaction is secondary to Other causative factors within the tissue. There does appear to be one significant difference between keloidal and normal scars that is observable with the electron microscope. This difference is the documentation of myofibroblasts in normal scars and the general absence of such cells in this keloidal dermis. We have observed a small population of cells that has some of the features of myofibroblasts, such as conspicuous endoplasmic reticulum in combination with abundant cytoplasmic microfilaments. However, other structural features as outlined by Cabbiani et al.'i are absent. These include attachment sites at the plasmalemma, differentiations of the cell surface, and the accordion-like morphology of the nucleus that results from cell contraction. It may be that the cells seen in the keloid were destined to become myofibroblasts, but never developed to full potential. There is evidence that myofibroblasts are responsible for contraction of granulation tissue that subsequently leads to normal scar formation.•• Myofibroblasts are also found in fibrocontractive diseases,'- and it is likely that myofibroblasts are present in the delayed contractions associated with hypertrophic scars. However, such contractions are not characteristic of keloid and our study suggests that functional myofibroblasts may be absent from this tissue. If such is the case, this would support the general concept that wound contraction may be the result of myofibroblast activity,'•'• If myofibroblasts develop normally and are present in significant numbers at the appropriate time, normal scars may be formed. If the development of a myofibroblast population is retarded, scar expansion may continue unchecked until cell differentiation is complete, resulting in delayed scar contraction as
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seen in the hypertrophic scar. In the keloid, it may be that myofibroblast development is abortive and not capable of contributing to scar resolution; therefoie, a massive overgrowth is possible. Such thoughts are clearly speculative, and the development of experimental models of all types of excessive scar formation is vital to an understanding of these pathologic processes. One other ultrastructural observation that deserves comment is the unexpected finding of intact nerves in the keloidal dermis. This was not anticipated because the patient had lepromatous leprosy, and usually the peripheral nerve processes in the skin of such patients are destroyed. The role of nerve processes in scar formation is not clear, but it may be that viable nerves are necessary not only for the development of normal scars, but for keloids and hypertrophic scars as well. If such a relationship does exist, this might partially explain the development of keloids in this patient, and also help to explain the rarity of keloid formation in other patients with lepromatous leprosy that possess denervated skin. Acknowledgment: The authors tbank Mrs, Janell Buck for ber tecbnical assistance, Mr. Carbis Kerimian for bis pbotograpbic assistance, and Mrs. Eunice Schwartz for ber secretarial services.
References 1, Coben, I, K,, Keiser, H, R,, and Sjoerdsma, A,, Collagen syntbesis in buman keloid and hypertropbic scar, Surg, Forum 22:488, 1971.
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2, Linares, FH, A,, Kiscber, C, W,, Dobrkowsky, M,, and Larson, D. L,, Tbe bistiotypic organization of tbe bypertropbic scar in bumans. J, Invest. Dermatol, 59:323, 1972, 3, Ten Cate, A, R,, and Freeman, E,, Collagen remodelling by fibroblasts in wound repair. Preliminary observations, Anat, Rec, 179: 543, 1974, 4, Ryan, C, B,, Cliff, W, J,, Cabbiani, C, Irle, C, Montandon, D,, Statkon, P, R,, and Majno, C , Myofibroblasts in human granulation tissue. Hum, Pathol, 5:55, 1974, 5, Ketcbum, L, D,, Coben, I, K,, and Masters, F, W,, FHypertropbic scars and keloids, A collective review, Plast, Reconstr, Surg, 53: 140, 1974. 6, Peacock, E, E,, Madden, J, W,, and Trier, W, C, Biologic basis for tbe treatment of keloids and bypertropbic scars, Soutb, Med. J, 63:755, 1970, 7, Kiscber, C, W,, Linares, H,, Dobrkowsky, M,, and Larson, D, L,, Electron microscopy of tbe byperlropbic scar. In: 29tb Ann, Proc, Electron Microscopy Soc, Amer, Edited by Arceneaux, C, J, Boston, 1971, pp. 302-303. 8, Kiscber, C, W,, Fibroblasts during bypertrophic scar formation and resolution. 31st: Ann, Proc, Electron Microscopy Soc, Amer. Edited by Arceneaux, C, J, New Orleans, 1973, pp, 428-429, 9, Kischer, C, W,, Collagen and dermal patterns in hypertropbic scar, Anat, Rec, 179:137, 1974. 10, Ross, R,, and Odiand, C , Human wound repair, II, Inflammatory cells, epitbelialmesencbymal interrelations, and fibrogenesis, J, Cell Biol, 39:152, 1968, 11, Cabbiani, C , Ryan, C, B,, and Majno, G., Presence of modified fibroblasts in granulation tissue and tbeir possible role in wound contraction, Experientia 27:549, 1971, 12, Cabbiani, C , and Majno, C , Dupuytren's contracture: fibroblast contraction? An ultrastructural study. Am, J, Patbol, 66:131, 1972, 13, Madden, J, W,, Morton, D , and Peacock, E, E,, Contraction of experimental wounds. I, Inhibiting wound contraction by using a topical smootb muscle antagonist. Surgery 76:8, 1974,
Against ALOPECIA AREATA, Eb 782 recommends the dirt of flies, or the dirt under the finger nails.—Chaliounqui, P.: Magic and Medical Science in Ancient Egypt. London, Hodder and Stoughton, 7963, p. 137.
ULTRASTRUCTURE OF KELOID: AN UNUSUAL INCIDENT INVOLVING LEPROMATOUS LEPROSY ROBERT F, DYER, Ph,D,, AND CARL D, ENNA, M,D, From the Department of Anatomy, Louisiana State University Medical Center, New Orleans, and the Department of Surgery, USPHS Hospital, CarviUe, Louisiana
ABSTRACT: A patient w/fh lepromatous leprosy developed keloids on the dorsum of both arms in response to ulcerations due to acute erythema nodosum leprosum reactions. Electron m/croscop/c exam/nation of the keloidal dermis showed a morphology indicative of increased production of normal collagen fibrils. The greatest cellular changes from normal were in fibroblasts which were enlarged due to increased amounts of rough endoplasmic reticulum and extensive Colgi complexes. Nuclear folds were also evident in these fibroblasts. Some cells, considered to be fibroblasts, were filled with cytoplasmic fifamehts and contained bizarre shaped nuclei. Mast cells, blood vessels and nerve processes were also present.
lepromatous leprosy usually form scars that are flat, atrophic and nonadherent, apparently due to the disruption and destruction of connective tissue elements by the disease infiltrate. However, a patient at the USPHS Hospital at Carville, Louisiana, did manifest keloids on the posterior aspect of the upper arms in response to ulcerations caused by acute erythema nodosum leprosum reactions. Since biopsy specimens were necessary for diagnosis, we had the opportunity to investigate the ultrastructure of a condition that rarely occurs in leprosy patients. In this article we will discuss tine ultrastructural features of keloid in general, and specifically in relation to this leprosy patient.
Keloids and hypertrophic scars develop in traumatized human skin when the normal equilibrium between collagen synthesis and degradation is disrupted. Biochemical studies have shown that this disruption favors excessive collagen synthesis by fibrobiasts of the involved derm i s . i Ultrastructural studies of hypertrophic scars support the biochemical evidence, because the majority of fibroblasts depict a morphology indicative of h i g h rates of protein synthesis.^ A similar ultrastructurai morphology probably exists for keloids, but electron microscopic studies of this type of scar are lacking.
Materials and Methods The patient, a 21-year-old Caucasian man with active lepromatous leprosy, was admitted to the USPHS Hospital at Carville. Ulcerations due to acute reactions developed on the dorsal aspect of both arms, and the healing process resulted in excessive scar formation that continued to enlarge with time. Biopsies
Keloids rarely form in patients with lepromatous leprosy, even though the denervated skin of these patients is exposed to frequent injury. Patients with
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of the scars were removed approximately 18 months after the initial ulcerations, and the diagnosis of keloid was confirmed after gross and light microscopic examination. Portions of the scar tissues were also prepared for electron microscopy. Fresh samples were placed in cold 4% glutaraldehyde buffered to pH 7.2 with 0.2 m phosphate buffer, fixed for at least 24 hours, and rinsed in 0.2 m phosphate buffer containing 0.3 m sucrose. The dermis and epidermis were separated, and the dermal tissue was diced into 1 mm cubes and post-fixed for 2 hours in 1% osmium tetroxide buffered to pH 7.2 with 0.2 m phosphate buffer. The samples were dehydrated in a graded series of alcohols, equilibrated with propylene oxide, and embedded in Spurr embedding medium. Thin sections were cut on a Reichert OMU2 ultramicrotome, stained with lead and uranyl ions, and viewed with a Philips EM 200 electron microscope. Results
The morphology described is restricted to the reticular layer of the keloidal dermis. A sample of nonkeloidal dermis from an adjacent site did not reveal any of the unusual ultrastructural features characteristic of the keloid. Therefore, it is assumed that the results reflect dermal changes related to keloid formation rather than to lepromatous leprosy. Figure 1 shows that the structure and organization of connective tissue cells and fibers were disrupted in the keloidal dermis. Fibroblasts were enlarged, and collagen closely invested all cellular elements, obliterating spaces normally seen between collagen bundles and around cells. Collagen fibrils were usually ori-
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ented in the same direction, but deviations from this parallel arrangement occurred, especially adjacent to connective tissue cells (Figs.^2, 5, 8). The fibrils approximated 1000 A in diameter, had a normal periodicity of 640 A, and interfibriilar matrix material was scant. Elastic fibers were not demonstrated in the keloidal dermis, a condition that is characteristic of keloids in general. Three categories of connective tissue cells were obvious in the keloidal dermis: (1), cells comprising the fibroblast population, (2), cells of the macrophage system, and (3), the mast cell population, usually associated with neurovascular structures. The fibrobiast population was composed of a number of cell types that are considered to reflect different functional states of the same cell. One type had the structure of fibrocytes. These cells were flattened, had an attenuated cytoplasm with few organelles, and a smoothsurfaced nucleus that conformed to the cell outline. The nucleus sometimes contained a well-developed nucleolus (Fig. 2). Another cell type in the first category was identifiable as very active fibroblasts. These cells were the predominant cell population in the keloid dermis, and they were enlarged by accumulations of rough endoplasmic reticulum and Colgi complexes (Fig. 3). Nuclear volume also appeared increased, although changes in cell shape could have accounted for this impression in a single plane of section. More obvious changes were projections and indentations of the nuclear envelope (Fig. 3), in contrast to the smooth-surfaced nuclei of fibrocytes (Fig. 2). Higher magnification of the active fibroblast cytoplasm showed large Golgi
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Fig. 1. Vievi' of reticular layer of keloidal dermis showing a fibroblast (F) and portions f other cells embedded in a dense collagen matrix (C) ( x 5000).
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Fig. 2. Top, a fibrocyle exhibiting a smooth surfaced nucleus (N) containing a prominent nucleolus (Nu). Collagen (C) is seen in different planes of orientation (X 2600). Fig. 3. Bottom, a fibroblast enlarged by numerous Golgi complexes (G) and cisternae of rough endoplasmic reticulum (ER). The nucleus (N) has an irregular surface (X 7500).
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Fig. 4. Top, high magnificalion of fibroblasi (yU)|)l,isni showing a Golgi zone (C) composed of vacuoles (V) and smaller vessels (VE), Microfilaments (MF) and microtubules (arrows) are seen, (ER, rough endoplasmic reticulum) (x 12,500), Fig, 5, Bottom, fibroblast stained with phosphotungstic acid. Collagen is seen extracellulary in different planes of section (C) and also the cytoplasm of the fibroblast (C with arrows) (X 12,500).
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complexes consisting of numerous vacuoles and vesicles (Fig. 4). Tbe larger vacuoles had no visible content, some medium-size vacuoles contained a flocculent precipitate, and the smaller vesicles were a heterogeneous population that included coated vesicles. The profiles of rougb endoplasmic reticulum, although numerous, were generally not dilated, and the cisternae contained a precipitate similar to tbat of the Colgi vacuoles (Fig. 4). The cytoplasm of tbese cells also had variable amounts of microfilaments and a few microtubules (Figs. 4, 5). In addition, fibroblasts sometimes contained collagen fibrils witbin their cytoplasm (Fig. 5), a feature of fibroblasts involved in collagen remodelling associated with wound repair.^ Certain cells witbin the keloid had an unusual morphology, but were still considered components of the fibroblast population. They contained pleomorpbic nuclei and large amounts of rougb endoplasmic reticulum, but their predominant cytoplasmic feature was an abundance of randomly organized microfilaments (Fig. 6). Otber cells of this type contained a larger population of microfilaments that were more organized in appearance (Fig. 7). Tbese cells contained fewer cytoplasmic organelles, but distention of rough endoplasmic reticulum was a dominant morphological feature. In other examples of this peculiar cell type (Fig. 8), the nucleus was large and pleomorphic, with a well-developed nucleolus. The cytoplasm was scant relative to other fibroblast cells and mostly occupied by a feltwork of microfilaments. Tbe organization of the microfilaments and the presence of small densities along their length was similar to that seen in myofibroblasts of contracting granulation tissue;"* however, other features of tbe myofibroblast are not
December 1975
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evident, and tbese cells were considered a part of the fibroblast population. The general category of cells, wbich were components of tbe macrophage system, were considerably fewer than the active fibroblasts. The cytoplasm of the macrophages contains scattered profiles of rough endoplasmic reticulum and some microfilaments. Lysosomes and phagocytic vacuoles were only occasionally present in tbe peripheral cytoplasm, but an obvious cytoplasmic feature of many macrophages was a vacuolization in tbe region of tbe nucleus (Figs. 9, 10). These vacuoles varied in size and content; some appeared clear, others contained a homogeneous material, and others contained membranous whorls (Fig. 10). Occasionally the nuclei of the macrophages contained nuclear inclusions (Fig. 9). Tbe third category of cells, the mast cells, occurred in large numbers in association with blood vessels and accompanying nerves tbat coursed through the keloidal dermis (Fig. 11). The cells contained granules that exhibited a homogeneous internal structure, or a crystalline or scroll-like membrane pattern. Degranulated mast cells were rarely seen in the keloidal dermis. Small blood vessels and related pericytes were common in keloidal dermis (Fig. 11), but the lumina were often collapsed and red cells were infrequent. Blood vessels were anticipated in the tissue, but nerve processes were not; however, in this case, small intact nerve bundles were usually found in the company of mast cells and blood vessels. Usually tbese nerve bundles consisted of unmyelineated axons with an associated Schwann cell (Fig. 11). These bundles were frequently embedded in a collagen fibril matrix, which in turn was invested by a connective tissue cell (Fig. 11).
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Fig. 6. Type of fibroblast containing an irregular nucleus (N) and many unorganized microfilaments (MF) (X 26,600).
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Fig. 7. Top, type of fibroblast exhibiting a pleomorphic nucleus (N) and distended cisternae of rough endoplasmic reticulum (ER), Bundles of microfilaments (MF) occupy the cytoplasm ( x 19,200), Fig, 8, Bottom, fibroblastic cell with some characteristics of a myofibroblast. Small densities which are similar to contraction bands are seen at the arrows. (N, nucleus; Nu, nucleolus; C, collagen) ( x 7500).
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Fig. 9. Top, cell identified as a macrophage. Accumulations of cytoplasmic vacuoles (CV) are seen in close relationship to the nucleus (N), (NI, nuclear inclusions) (X 4500), Fig. 10. Bottom, macrophage exhibiting cytoplasmic vacuoles (CV) surrounded by the nucleus (N). The fibrous lamina of the nucleus (FL) and the associated outer leaflet of the nuclear envelope completely surround the vacuoles (X 9900).
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Fig. 11. Top, mast cells (MC) in company with a small blood vessel and nerve, Tbe lumen of the vessel (L) is collapsed and pericytes (P) are seen, Tbe nerve bundle (NB) is surrounded by collagen (C) and a connective tissue cell (CTC), (E, endotbelial cell) ( x 4500), Fig. 12. Bottom, a single axon (AX) enveloped by its Scbwann cell (SC) ( x 17,000).
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Other nerve processes were found at a distance from the vascular network, but these were usually single axons that contained mitochondria and many neurofilaments. These axons were frequently enveloped by a Schwann cell (Fig. 12). Discussion The gross and the histologic appearance of keloids are well documented,'' b u t there is controversy over the classification of this type of excessive scar. However, most investigators consider keloids and hypertrophic scars to be manifestations of the same process.2' ^ Hypertrophic scars have been studied a n d characterized with the electron microscope,-' ""' but to our knowledge similar studies on keloids, particularly in patients with leprosy, are not available. Although our study is limited to mult i p l e lesions from a single patient, it is possible to state that the electron microscopic features of these keloid lesions d o not differ significantly from those of hypertrophic scars. It may be of greater importance to contrast the structure of keloids and hypertrophic scars to normal scars. The gross differences are obvious, and the histologic features of excessive scars and normal scars permit distinctions to be made, but neither of these levels of observation provide a clear insight into factors responsible for excessive scar formation. Biochemical and ultrastruct u r a l studies indicate that excessive scars are a result of increased collagen production by hypertrophied fibroblasts that have probably also undergone hyperplasia. This fibroblastic response is not unique to keloids and hypertrophic scars, since a similar response occurs in normal scar formation.'" However, the response is maintained for abnormal periods of time in those instances in which excessive scars are formed. The fibroblasts produce the fibrous elements that
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result in massive scar formation, but it may be that this reaction is secondary to Other causative factors within the tissue. There does appear to be one significant difference between keloidal and normal scars that is observable with the electron microscope. This difference is the documentation of myofibroblasts in normal scars and the general absence of such cells in this keloidal dermis. We have observed a small population of cells that has some of the features of myofibroblasts, such as conspicuous endoplasmic reticulum in combination with abundant cytoplasmic microfilaments. However, other structural features as outlined by Cabbiani et al.'i are absent. These include attachment sites at the plasmalemma, differentiations of the cell surface, and the accordion-like morphology of the nucleus that results from cell contraction. It may be that the cells seen in the keloid were destined to become myofibroblasts, but never developed to full potential. There is evidence that myofibroblasts are responsible for contraction of granulation tissue that subsequently leads to normal scar formation.•• Myofibroblasts are also found in fibrocontractive diseases,'- and it is likely that myofibroblasts are present in the delayed contractions associated with hypertrophic scars. However, such contractions are not characteristic of keloid and our study suggests that functional myofibroblasts may be absent from this tissue. If such is the case, this would support the general concept that wound contraction may be the result of myofibroblast activity,'•'• If myofibroblasts develop normally and are present in significant numbers at the appropriate time, normal scars may be formed. If the development of a myofibroblast population is retarded, scar expansion may continue unchecked until cell differentiation is complete, resulting in delayed scar contraction as
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seen in the hypertrophic scar. In the keloid, it may be that myofibroblast development is abortive and not capable of contributing to scar resolution; therefoie, a massive overgrowth is possible. Such thoughts are clearly speculative, and the development of experimental models of all types of excessive scar formation is vital to an understanding of these pathologic processes. One other ultrastructural observation that deserves comment is the unexpected finding of intact nerves in the keloidal dermis. This was not anticipated because the patient had lepromatous leprosy, and usually the peripheral nerve processes in the skin of such patients are destroyed. The role of nerve processes in scar formation is not clear, but it may be that viable nerves are necessary not only for the development of normal scars, but for keloids and hypertrophic scars as well. If such a relationship does exist, this might partially explain the development of keloids in this patient, and also help to explain the rarity of keloid formation in other patients with lepromatous leprosy that possess denervated skin. Acknowledgment: The authors tbank Mrs, Janell Buck for ber tecbnical assistance, Mr. Carbis Kerimian for bis pbotograpbic assistance, and Mrs. Eunice Schwartz for ber secretarial services.
References 1, Coben, I, K,, Keiser, H, R,, and Sjoerdsma, A,, Collagen syntbesis in buman keloid and hypertropbic scar, Surg, Forum 22:488, 1971.
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2, Linares, FH, A,, Kiscber, C, W,, Dobrkowsky, M,, and Larson, D. L,, Tbe bistiotypic organization of tbe bypertropbic scar in bumans. J, Invest. Dermatol, 59:323, 1972, 3, Ten Cate, A, R,, and Freeman, E,, Collagen remodelling by fibroblasts in wound repair. Preliminary observations, Anat, Rec, 179: 543, 1974, 4, Ryan, C, B,, Cliff, W, J,, Cabbiani, C, Irle, C, Montandon, D,, Statkon, P, R,, and Majno, C , Myofibroblasts in human granulation tissue. Hum, Pathol, 5:55, 1974, 5, Ketcbum, L, D,, Coben, I, K,, and Masters, F, W,, FHypertropbic scars and keloids, A collective review, Plast, Reconstr, Surg, 53: 140, 1974. 6, Peacock, E, E,, Madden, J, W,, and Trier, W, C, Biologic basis for tbe treatment of keloids and bypertropbic scars, Soutb, Med. J, 63:755, 1970, 7, Kiscber, C, W,, Linares, H,, Dobrkowsky, M,, and Larson, D, L,, Electron microscopy of tbe byperlropbic scar. In: 29tb Ann, Proc, Electron Microscopy Soc, Amer, Edited by Arceneaux, C, J, Boston, 1971, pp. 302-303. 8, Kiscber, C, W,, Fibroblasts during bypertrophic scar formation and resolution. 31st: Ann, Proc, Electron Microscopy Soc, Amer. Edited by Arceneaux, C, J, New Orleans, 1973, pp, 428-429, 9, Kischer, C, W,, Collagen and dermal patterns in hypertropbic scar, Anat, Rec, 179:137, 1974. 10, Ross, R,, and Odiand, C , Human wound repair, II, Inflammatory cells, epitbelialmesencbymal interrelations, and fibrogenesis, J, Cell Biol, 39:152, 1968, 11, Cabbiani, C , Ryan, C, B,, and Majno, G., Presence of modified fibroblasts in granulation tissue and tbeir possible role in wound contraction, Experientia 27:549, 1971, 12, Cabbiani, C , and Majno, C , Dupuytren's contracture: fibroblast contraction? An ultrastructural study. Am, J, Patbol, 66:131, 1972, 13, Madden, J, W,, Morton, D , and Peacock, E, E,, Contraction of experimental wounds. I, Inhibiting wound contraction by using a topical smootb muscle antagonist. Surgery 76:8, 1974,
Against ALOPECIA AREATA, Eb 782 recommends the dirt of flies, or the dirt under the finger nails.—Chaliounqui, P.: Magic and Medical Science in Ancient Egypt. London, Hodder and Stoughton, 7963, p. 137.
