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Evidence of remodeling in dura mater cardiac valves
J THoRAc
CARDIOVASC SURG
84:267-281, 1982
Evidence of remodeling in dura mater cardiac valves Subcellular changes in 12 dura mater cardiac valves (in the mitral or aortic position) surgically removed after 23 to 108 months of implantation owing to calcification, rupture, or endocarditis show signs of a remodeling process. Significant morphologic changes in the connective tissue fiber matrices and cell populations were noted in the recovered valvular leaflets. Macrophages were found within electronlucent (cleared-out) areas, and they seemed to play an essential role in the remodeling process by ingesting and digesting selected connective tissue components. Fibroblasts found within these "rebuilding" areas in the dura mater tissue possessed small cytoplasmic vesicles (65 nm in diameter) being extruded from the cell. Evidence of early collagen formation was also found in association with both peripheral filaments and peripheral condensations, as well as within the connective tissue matrices surrounding the cellular elements, where electron dense amorphous material was observed. In conclusion, the long-term durability of dura mater bioprosthetic cardiac valves may be directly related to ( J) glycerin stabilization and preservation of the collagen fibers, (2) the viability of the fibroblasts and macrophages within the implanted valves. and (3) the unique morphology and fine structure of the double-layered dura mater encephali . We hypothesize that the fibroblasts or myofibroblast-like cells found within the implanted leaflets. no matter what their origin. are capable of giving form and organization to the early developing connective tissue.
Delmas J. Allen, Ph.D., Toledo, Ohio, Gregory J. Highison, Ph.D.,** Reno, Nevada, Liberato 1. A. DiDio, M.D., Ph.D.,* Toledo, Ohio, E. J. Zerbini, M.D.,*** and L. B. Puig, M.D.,*** Sao Paulo, Brazil
Since the first implantation of biological valve prostheses in man, )-3 various types of tissue, including autologous and homologous fascia lata, heterologous pericardium, porcine heterografts, and homologous dura mater, have been used to replace defective human cardiac valves. As these valvular devices were functionally silent, nonhemolytic, and nonthrombogenic, the initial results obtained with bioprosthetic cardiac valve implantations were encouraging, but the durability of these valves has been questioned. The durability or long-term resistance to mechanical wear of the early
Address for reprints: Dr. Delmas J. Allen, Department of Anatomy, Medical College of Ohio, C.S. 10008, Toledo, Ohio 43699.
bioprosthetic cardiac valves was considered to be a function of host fibroblast infiltration and deposition of new connective tissue fibers. 4-6 Carpentier and associates? refuted the existing hypothesis that viability of the cardiac bioprosthesis was essential to long-term durability by demonstrating that host cell ingrowth was often more harmful than beneficial to the implanted cardiac bioprosthesis. They observed very little new collagen formation or host fibroblast infiltration in the aortic heterografts. Instead, Carpentier and colleagues 7 found a predominant host inflammatory cell infiltration leading them to conclude that long-term durability was dependent upon (1) prevention of host inflammatory cell infiltration, (2) reduction in the antigenicity of the graft, and (3) stabilization of the collagen fiber matrix with intramolecular cross-linkages. These postulates have since been widely accepted and confirmed by the proved long-term durability of the glutaraldehyde-treated porcine heterograft. At the ultrastructural level, the nonviability of the glutaraldehyde-treated porcine heterograft was demonstrated by Ferrans and associates. 8, 9 After noticing that
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From the Departments of Anatomy, Medical College of Ohio and University of Nevada School of Medical Sciences, and the Heart Institute, University of sao Paulo, sao Paulo, Brazil. Supported by grants to Dr. D. J. Allen from the American Heart Association, Northwestern Ohio Chapter, Inc., and the National Institutes of Health (BRS Grant 5-S01-RR-05700-II). Received for publication Sept. II, 1981. Accepted for publication Nov. 24, 1981.
0022-5223/82/080267+ 15$01.50/0
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Table I. Case data Case No. I
2
3 4 5 6 7 8 9 10 II
12
Age (yr)
31 29 44 7 18 33 48 57 36 50 29 37
Sex
Time of implantation (mo)
Valve location
Cause for reoperation
F F M M F F M M M M M M
97 100 59 23 67 50 70 66 108 27 90 66
Mitral Mitral Aortic Mitral Mitral Aortic Aortic Aortic Mitral Aortic Aortic Aortic
Calcification Calcification Rupture Calcification Rupture Rupture Rupture Rupture Rupture Rupture Endocarditis Rupture
progressive breakdown of collagen occurred in heterografts implanted longer than 2 months, they concluded that such breakdown was the crucial factor in determining the long-term durability of the glutaraldehydetreated porcine bioprosthesis, a deterioration that had not been reported by previous gross anatomic and histologic examinations.!? A comparison between the findings by Ferrans and associates" on the glutaraldehydetreated porcine heterograft and our" observations on the glycerin-treated dura mater allografts revealed that both bioprosthetic valves have equivalent long-term durability records, and the clinical success of both valves has been attributed to specific preimplantation treatment. Postimplantation surface alterations observed in the study of glycerin-treated dura mater valves were similar in many aspects to those described by Ferrans' group" for the glutaraldehyde-treated porcine heterografts. Surface fibrin formation appeared to be a natural response of vascular prostheses in general. However, a comparison of the cell types found within the glycerin-treated dura mater allografts and glutaraldehyde-treated porcine heterografts showed significant differences. In direct contrast, macrophages and fibroblasts were found throughout the implanted dura mater allografts. The primary objective of this investigation is to present subcellular or ultrastructural changes in dura mater valvular leaflets of a remodeling process which appears to be unique to this particular valvular prosthesis. Material and method
The twelve dura mater cardiac valves examined in this study were constructed, implanted into patients, and recovered at operation by Zerbini'" and his collaborators at the Heart Institute of the University of Sao
Paulo, Brazil. The list of the cases is given in Table I. The bioprostheses had to be removed because of valvular dysfunction. Dura mater encephali was removed from cadavers of 10- to 50-year-old human donors within 20 hours after death. Subjects with infective or degenerative diseases, as well as neoplasia, were excluded. After being rinsed in a continuous flow of water for I to 2 hours, the recovered dura mater encephali was placed in a sterile container of 98% glycerin at room temperature for 20 to 30 days before being constructed into valvular leaflets. 13 The aortic dura mater allografts recovered at operation after various periods of implantation (23 to 108 months) were gently irrigated with 0.8% heparinized physiological saline solution before placement in 3% glutaraldehyde solution (3 parts glutaraldehyde and 97 parts 0.144N sodium cacodylate buffer, pH 7.4). Upon arrival at the laboratory of the Department of Anatomy at the Medical College of Ohio at Toledo, the allografts were placed in the appropriate fresh fixation solution. Representative tissue samples were dissected from (I) the ring and strut areas of the support frame, (2) midline, and (3) along the free edge of both recovered and control valvular leaflets. Dura mater recovered at necropsy, preserved in 98% glycerin but not formed into bioprosthetic valves, and nonimplanted, glycerin-preserved dura mater valves were used as controls. Representative samples from each of the leaflets were rinsed in O.I44N sodium cacodylate buffer (pH 7.4) and processed for transmission electron microscopy by standard methods as described by Highison and associates. II In order to demonstrate connective tissue elements, sections were cut and stained according to the protocol described by Snodgrass." Stained ultrathin sections were examined and photographed with either a Hitachi llE-l electron microscope at 75 k V or a Phillips 300 electron microscope at 80 k V. Images were recorded on Kodak SO-163 film. Results
At the subcellular level, significant morphologic differences in the connective tissue fiber matrices and cell populations were noted when the recovered valvular leaflets were compared with the controls (cadaver dura mater and nonimplanted valves). Both types of control tissue were relatively acellular, and collagen (fibrils within each of the two layers (endosteal and meningeal) appeared to be arranged into parallel arrays or lamellae. In the recovered leaflets, however, distinct lamellae
Volume 84 Number 2 August, 1982
Dura mater cardiac valves
Fig. 1. Area of remodeling. Electron micrograph of portions of several fibroblasts (arrows) among the connective tissue components of recovered dura mater valvular leaflets. Intracellular and extracellular fine filaments are also present. The more densely stained orcein-positive material was always extracellular (arrowheads). Note the fine filamentous (wispy) material throughout the connective tissue matrix. (Orcein stain, xS,OOO.)
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Fig. 2. Remodeling in recovered valve. Portions of the nuclei and surrounding cytoplasm of at least two elongated fibroblasts predominate in this electron micrograph. Cytoplasmic and membrane-associated vesicles (arrow) are discernible at this magnification. Intracellular fine microfilaments and extracellular orcein-stained material are seen. Note the granular chromatin material with only a thin band lining the nuclear envelope. An occasional onionoid body (corpus cepiforme) can be visualized (arrowhead). (Orcein stain, x7,500.)
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Dura mater cardiac valves
Fig. 3. A fibroblast in a remodeling area. Numerous perinuclear small cytoplasmic vesicles measuring approximately 65 nm in diameter are present. Microfilaments and microtubules run parallel to the long axis of the cell subjacent to the cell membrane. Peripheral (marginal) filaments often form streamer-like processes (arrow) which extend from the cell surface suggesting motility. (Orcein stain, xS,OOO.)
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Fig. 4. Ultrastructure of cytoplasmic microfilaments and vesicles. Cytoplasmic vesicles (arrow) were observed in clusters among the cytoplasmic microfilaments. The beaded nature of the orcein-positive material and the relationship to the fibroblast are apparent at this magnification . (Orcein stain, x 15,380.)
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August, 1982
were usually absent after 23 months of implantation; these leaflets were composed of numerous cells and cellular processes with collagen fibers interspersed among them (Figs. 1 to 3). The collagen fibrils were arranged in criss-crossing bundles, as was evident from longitudinal, transverse, and tangential sections of collagen noted in the same electron microscopic field (Fig. 1). In low-power electron microscopic fields, both orcein-positive structures and areas of reabsorption were observed. The fibroblasts within valves implanted for 23 months and longer possess dense areas in and around their periphery (Figs. 2 to 4). At low magnifications (Figs. 2 and 3), these areas of peripheral condensation appear either as smudges (less dense) near the surface of the cell (Fig. 2) or as dark-staining beads (Fig. 4). The peripheral condensations observed in these cells (Figs. 2 to 4) also reveal a less dense, fibrillar component that is contiguous with the endoplasm. A parallel arrangement of delicate, threadlike or filamentous material that comprises the "endoplasmic condensation" is discernible (Fig. 4). These filaments show no evidence of periodicity or striations. The cell boundaries of these fibroblasts are not limited or marked by a distinct plasmalemma. This loss of a definitive outer cell membrane appears to be a distinguishing morphologic characteristic of cells undergoing peripheral and endoplasmic condensation. Fibroblasts found within these "rebuilding" areas possessed small cytoplasmic vesicles (Figs. 3 and 4) measuring 65 nm in diameter; these often were located in a peripheral position, suggesting that they were in the process of being extruded from the cell. Remodeling and turnover of connective tissue involve both the formation and degradation of collagen. The best evidence of early collagen formation in dura mater valves is found in association with peripheral filaments and peripheral condensation. Collagen fibrils, usually only a few bands in length, are found beside and seemingly within the peripheral filaments and condensations (Fig. 4). These short fibrils are usually amorphous, but they sometimes appear beaded. In accordance with present knowledge of connective tissue synthesis, the presence of the amorphous material with this positional relationship suggests that it contains precursors of collagen. Peripheral filaments may also form streamer-like extensions (Fig. 3) which project or trail from the fibroblasts, suggesting motility. In light microscopy, similar processes have been observed and referred to as stress fibers, tonofibrils, and fibroglia. Additional evidence for the formation or synthesis of collagen was found within the connective tissue matrices surround-
ing the cellular elements where electron-dense amorphous material was observed (Fig. 5). Finely granular and beaded fibrils with a cross-banding periodicity of 63 to 65 nm (the standard periodicity of collagen) between beads were located at the periphery of the amorphous component. Areas within the valvular leaflets where active degradation was adjudged to be occurring were characterized by the presence of lipid infiltration (Fig. 6) and macrophages (Figs. 7 to 9). Within these areas of remodeling and turnover of connective tissue fiber components, foci of progressive degradation were observed and characterized by (I) a decrease in the density of the collagen matrix, presenting with cleared-out areas (Fig. 7) and macrophages, (2) an increase in the extracellular fine filaments and granular material (Figs. 7 and 8), (3) lipid deposition, and (4) variability in the staining properties of the connective tissue ground substance and fibers. Macrophages were often observed within "cleared-out" or electron-lucent areas of the leaflets, believed to be caused by their phagocytosis and degradation of collagen (Fig. 7). These electron-lucent areas were usually surrounded by erratically staining connective tissue and ground substance. The macrophages possessed prominent multilobulated nuclei with heterochromatin located at the periphery of the nuclear membrane, few mitochondria and lysosomes, numerous clear and dense membrane-bound vesicles, and onionoid bodies in different stages of development or dense myelin-like figures." These phagocytic cells appeared singly and in small groups, but most frequently in large groups constituting nests (Fig. 9).
Discussion Since these were the only valves that needed surgical reimplantation, these studies are restricted so far to dura mater valves implanted for a minimum of 23 months. However, the data already obtained provide evidence of remodeling and turnover of connective tissue as revealed by active synthesis and degradation of collagen. Foremost among the observations reported herein are (1) that collagen synthesis appears to be related to certain morphologic characteristics of closely associated fibroblasts and (2) that collagen degradation occurs in or near areas where numerous active phagocytic cells were observed. The presence of either peripheral condensation or peripheral filaments in close proximity to both fibroblasts (regarded to be actively producing collagen) and mature collagen fibers was interpreted as evidence for collagen synthesis. In addition, the unique staining characteristics and special
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Fig. 5. Orcein-positive area within the connective tissue matrix. These orcein-positive areas within recovered valvular leaflets commonly demonstrated a centrally positioned amorphous component surrounded by fine granular and "beaded" material. The darker beads occurred at intervals averaging 65 nm. Thin sections treated with silver stain produced identical staining properties to those stained with orcein. (x 13,200.)
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Dura mater cardiac valves
August. 1982
Fig. 6. Lipid infiltration in recovered valve. A higher magnification of a selected area within a remodeling area revealed fenestrated or reticulated patterns (lipid droplets) and foci of variability in affinity for the orcein stain (darker areas). The connective tissue fibers in these areas also demonstrated a wide range in size. (Orcein stain, x 10,000.)
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Fig. 7. Macrophage within a cleared-out portion of a remodeling area . Macrophages, with numerous small plasmalemmal extensions similar to the one shown here , were observed within electron-lucent (cleared-out) areas . They were frequently surrounded by unevenly stained connective tissue fibers and a densely stained ground substance . (Uranyl acetate/lead citrate stain, x 14,000 .)
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August, 1982
Fig. 8. Transmission electron microscopic study of two macrophages located within connective tissue matrix of a recovered leaflet. The multilobulated nucleus with peripherally located dark-staining chromatin and fine plasmalemmal extensions are predominant morphologic traits of these cells. No cleared-out (electron-lucent) area surrounds these macrophages . (Uranyl acetate/lead citrate stain, x 13,200.)
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Fig. 9. A nidus of macrophages within a recovered leaflet. All of these cells possessed a multilobulated nucleus, Iysosomes, and residual or dense bodies . Dense amorphous material (left , middle) frequently appeared among these intrinsic cells and other connective tissue components . Intracellular and extracellular onionoid bodies (corpora cepiformia) are seen in different stages of development (arrows) . ( X 14.600 .)
Surgery
Volume 84 Number 2 August, 1982
affinity of the filaments, fibrils, ground substance, and amorphous components for lead citrate/uranyl acetate, silver, and orcein stains provide further support for the synthesis of connective tissue fibers. In general, elastic fibers readily stain with orcein, whereas reticular fibers stain with both orcein and ammoniacal silver. Since only negligible amounts of elastic and reticular fibers were observed in our light and electron microscopic studies of dura mater and recovered dura mater valves, the term "orcein positive" is utilized herein to refer to precursors of collagen and new collagen. The densely staining beaded strands found throughout areas of remodeling are interpreted as newly formed acid-mucopolysaccharide-covered collagen fibrils rather than elastic fibers. Control dura mater leaflets were relatively acellular when compared with those of implanted valves. In areas of remodeling, considered to be sites where collagen synthesis was taking place, both active and inactive fibroblasts were identified, whereas in foci demonstrating degradation of collagen fibers, cells with ultrastructural characteristics of active macrophages predominated. No evidence of a degradative role for the fibroblast in the remodeling and turnover of collagen in dura mater valves was noted, as reported in soft connective tissue by others. 16 Many questions have emerged as a result of the findings reported herein. I. Did the fibroblasts found within the remodeling areas originate from the surrounding cardiac tissues or from the circulating blood of the host, or were they of dural origin, having survived the preimplantation processing? 2. Are the peripheral condensation and peripheral filaments part of the exoplasm of the fibroblasts, or are they secretory products of these cells? 3. A similar question could be asked about the macrophages found within the implanted leaflets, including a possible interconversion between fibroblast and macrophage.lv !" 4. Is the remodeling observed within the implanted allografts part of a normal repair mechanism initiated by the continuous wear and tear on the leaflets, or is it a degeneration/repair sequence in response to a pathological stimulus? 5. How is degeneration related to preservation and/or viability of implanted tissue? It is evident from the electron microscopic findings reported herein that significant morphologic changes occurred within the connective tissue substance of long-term (23 to 108 months) implanted dura mater
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allografts when compared to the control tissue. The implanted dura mater leaflets are definitely not biologically inert, as previously assumed. Viable cells (macrophages and fibroblasts) observed within the 12 dural valves investigated in this study appear to be responsible for and in the process of conducting phagocytosis and synthesis (replacement) of the fiber matrix of the collagenous connective tissue. Thus the long-term durability of dura mater bioprosthetic cardiac valves may be directly related to (I) glycerin stabilization and/or preservation of the collagen fibers, (2) the viability of the fibroblasts and macrophages within the implanted valves, and (3) the unique morphology and fine structure of the double-layered dura mater encephali. At present, since we have not had dura mater valves implanted for less than 23 months available for study, we can only hypothesize that the fibroblasts found within the implanted leaflets, no matter what their origin, are capable of at least giving form and organization to the early developing collagen fibrils. The peripheral filaments may be related or correspond to the strands of mucinous material pulling off the surface of fibroblasts, as described by Porter and Pappas'" in the chick. They demonstrated that this "mucin" at the fibroblast surface was amorphous. In light of our observations and what is known about connective tissue formation, it seems reasonable to postulate that amorphous components associated with the fibroblasts within the dura mater valves possessed collagen precursors. Since this amorphous material was also observed to have a certain affinity for ammoniacal silver stain (which is known to stain reticular fibers and acid-mucopolysaccharides), the material may be interpreted as having some relationship to the argyrophilia of "immature" or "developing" collagen (reticulin fibrils). In fact, many argyrophilic fibers often appear near and seem to be associated with fibroblasts according to early light'"- 20 and transmission':' electron microscopic reports. However, as these':': 19.20 and our observations were limited to mateials fixed for either light or electron microscopy, the actual dynamics of collagen formation by fibroblasts and degradation by macrophages remain speculative. Evidence of remodeling (degeneration/synthesis of connective tissue fibers) in the dura mater valvular leaflets was observed in the connective tissue matrix of the dura mater tissue itself. Morphologic changes at the transmission electron microscopic level indicative ofthe remodeling process described herein were not seen either in the area of the' 'fibrous sheath" originating from the recipient or at the boundary of this sheath and the original dura mater tissue. The cells found within these
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areas where remodeling was observed possessed physicalor morphologic characteristics of either phagocytes (macrophages and polymorphonuclear leukocytes) or fibroblasts. These cellular elements were present in both control tissue and implanted dura mater valvular leaflets, but were more abundant in the latter. Although the fibroblasts or fibroblast-like cells found within both control and implanted leaflets showed some features intermediate between classical fibroblasts and smooth muscle cells (myocytes), they were not identified as myocytes primarily because (I) they appeared as single isolated cells, (2) they did not possess a well-developed lamellar (Golgi) complex, (3) they demonstrated the absence of a moderately developed granular endoplasmic reticulum, (4) they lacked numerous free ribosomes and consistent accumulations of glycogen particles typical of myocytes, and (5) there were no nerve fibers and terminals associated with these cells. Despite the extensive scanning and transmission electron microscopic studies on dura mater by Allen and Low, 21 Allen and DiDio,22 and Allen and associates;" cells with features typical of smooth muscle observed in other viscera" have not been reported in dura mater. In conclusion, the most important points derived from this investigation are (I) that glycerin-treated and preserved dura mater allografts are not biologically inert in the human circulation, (2) that dura mater valves implanted for significant periods of time show signs of collagen degeneration which are similar to those seen in other tissue valves, most notably the glutaraldehyde-treated heterograft valves, (3) that valvular cellularity appears to increase with time in the dura mater valves, unlike glutaraldehyde-treated valves, and (4) that the possibility exists that host cellular infiltrates into the dura mater valve may actively participate in the removal of destroyed collagen and in the production of significant amounts of new host collagen. REFERENCES Murray G: Homologous aortic-valve-segment transplants as surgical treatment for aortic and mitral insufficiency. Angiology 7:466-471, 1956 2 Beall AC, Morris GC Jr, Cooley DA, De Bakey ME: Homotransplantation of the aortic valve. J THoRAc CARDlOVASC SURG 42:497-506, 1961 3 Kerwin AS, Lenkei SC, Wilson DR: Aortic-valve homograft in the treatment of aortic insufficiency. Report of nine cases, with one followed for six years. N Engl J Med 266:852-857, 1962 4 Carpentier A: Utilisation d 'heterogreffes dans Ie traitement des lesions de I'appareil valvulaire aortique. Etude biologique et technique. Premiers resultats cliniques. These Med, Paris, 1966
Thoracic and Cardiovascular Surgery
5 Angell WW, Angell JD, Kosek JC: Clinical and experimental comparisons establishing the glutaraldehyde treated xenograft as the standard for tissue heart valve replacement. Tissue Heart Valves, Mllonescu, ed., London, 1979, Butterworth & Co., Ltd., pp 89-126 6 Angell WW, Buch WS, lben AB: Formalin preservation of porcine heterografts, Biological Tissue in Heart Valve Replacement, MI lonescu, DN Ross, RH Wooler, eds .. London, 1972, Butterworth & Co., Ltd., pp 543-552 7 Carpentier A, Lemaigre G, Robert L, Carpentier S, Dubost C: Biological factors affecting long-term results of valvular heterografts. J THORAC CARDIOVASC SURG 58:467-482, 1969 8 Ferrans VJ, Spray TL, Billingham ME, Roberts We: Structural changes in glutaraldehyde-treated porcine heterografts used as substitute cardiac valves. Transmission and scanning electron microscopic observations in 12 patients. Am J CardioI41:1159-1184, 1978 9 Ferrans VJ, Spray TL, Billingham ME, Roberts WC: Ultrastructure of Hancock porcine valvular heterografts. Pre- and post-implantation changes. Circulation 58:Suppl 1:10-18, 1978 10 Spray TL, Roberts WC: Structural changes in porcine xenografts used as substitute cardiac valves. Gross and histological observations in 51 glutaraldehyde preserved Hancock valves in 41 patients. Am J CardioI40:319-330. 1977 II Highison GJ, Allen OJ, DiDio LJA, Zerbini EJ, Puig LB: Ultrastructural morphology of dura mater aortic allografts after 44-73 months of implantation. J Submicros Cytol 12:165-187, 1980 12 Zerbini EJ: Results of replacement of cardiac valves by homologous dura mater valves. Chest 67:706-710, 1975 13 Zerbini EJ, Puig LB: The dura mater allograft valve. Tissue Heart Valves, M1 lonescu, ed., London, 1979, Butterworth & Co., Ltd., pp 253-30 I 14 Snodgrass MJ: Ultrastructural distinction between reticular and collagenous fibers with an ammoniacal silver stain. Anat Rec 187:191-205, 1977 15 DiDio LJA. The ubiquity of "myelin figures" as seen with electron microscopy. Acta Biomedica 43:341-406, 1972 16 Ten Cate AR, Deporter DA: The degradative role of the fibroblast in the remodelling and turnover of collagen in soft connective tissue. Anat Rec 182: 1-41, 1974 17 Kouri J, Ancheta 0: Transformation of macro phages into fibroblasts. Exp Cell Res 71: 168-176, 1972 18 Porter KR, Pappas GD: Collagen formation by fibroblasts of the chick embryo dermis. J Biophysic Biochem Cytol 5:153-166,1959 19 Maximow A: Development of argyrophile and collagen fibers in tissue cultures. Proc Soc Exp Bioi Med 25:439442, 1927 20 Wolbach SB: Controlled formation of collagen and reticulum. A study of the source of intercellular substance in recovery from experiment scorbutus. Am J Pathol 9:689-700, 1933
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21 Allen 01, Low FN: Scanning electron microscopy of the subarachnoid space in the dog. 1 Comp Neur 161:515540, 1975 22 Allen OJ, DiDio LJA: Scanning and transmission electron microscopy of the encephalic meninges in dogs. J Submicr Cytol 9: 1-22, 1977 23 Allen OJ, DiDio LJA, Highison GJ, Puig LB, Zerbini EJ:
The importance of scanning electron microscopy in surgery as seen by the study of recovered dural cardiac valve prostheses. Biomed Res 2: 253-264, 1981 24 Amenta F, Allen OJ, DiDio LJA, Motta P: A transmission electron microscopic study of smooth muscle cells in the ovary of rabbits, cats, rats and mice. J Submicr Cytol 11:39-51, 1979
Notice of correction
In the September, 1979, issue of the JOURNAL, an error was made in an article by D. Craig Miller and associates, entitled "Operative Treatment of Aortic Dissections: Experience With 125 Patients Over a Sixteen-Year Period" (78:365-382, 1979). In the original publication, the figure 33, which is enclosed in the box at the bottom of the tenth column of the table shown below, read 22. Retrospectively, the 22 referred to the number of deaths following medical treatment of acute type B aortic dissections and had not been converted into the percent (22/67 = 33%). Recent reference in other manuscripts has led to the discovery of the error and indicates the advisability of this belated correction. Table IX. Operative or hospital mortality and sample size in selected series since 1970 Acute Type B
Type A Surgical Series Maryland UCLA Yale PBBH* SUNY-Upstate THI-Houston:j: Alabama THI -Houstont Memphis THI-Houston:j: Virginia Average Present study
Chronic
Medical
Surgical
Medical
Surgical
Medical
Surgical
Medical
%
No·1 %
No·1 %
No·1 %
No·1 %
I
Reference
Year
No·1
%
No·1
%
No·1
%
No.
10 12 14 15 16 17 22 19 20 21 66
1971 1972 1974 1974 1975 1975 1976 1976 1976 1977 1979
10
60 89 45 23 100
5
100
50
80 78 50
19 6 2
21
17
88
22
24 8 51 26
24 21 63 18 73
50 50 42 75 0 25 36
8
15 9 2
10 2 12 8 I 49§ II
25
80
14
43
3
100
14
36
9
179 53
38 34
76
83
121 20
36 45
67
1979
9 9 22 3
17
Type B
Type A
12
67
10
40
0
9
0
32
13 25 15
15 8 7
0
4 42 10
0 17 40
5
0
14
14
6
50
18
28
74 29
18 14
15
20
72 23
25 22
23
22
Ot
100 67
@]
Legend: The data in the original reports have been reclassified when necessary to correspond with the Stanford type A and B classification criteria. *Peter Bent Brigham Hospital (>53% of 45 total cases were acute). tAli patients with acute type B dissections failing medical therapy crossed over to surgical therapy. :j:Texas Heart Institute; some patients in reference 21 are included in reference 19. §Six of 12 patients who died had retrograde extensions of their dissections in perioperative period.
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Evidence of remodeling in dura mater cardiac valves Subcellular changes in 12 dura mater cardiac valves (in the mitral or aortic position) surgically removed after 23 to 108 months of implantation owing to calcification, rupture, or endocarditis show signs of a remodeling process. Significant morphologic changes in the connective tissue fiber matrices and cell populations were noted in the recovered valvular leaflets. Macrophages were found within electronlucent (cleared-out) areas, and they seemed to play an essential role in the remodeling process by ingesting and digesting selected connective tissue components. Fibroblasts found within these "rebuilding" areas in the dura mater tissue possessed small cytoplasmic vesicles (65 nm in diameter) being extruded from the cell. Evidence of early collagen formation was also found in association with both peripheral filaments and peripheral condensations, as well as within the connective tissue matrices surrounding the cellular elements, where electron dense amorphous material was observed. In conclusion, the long-term durability of dura mater bioprosthetic cardiac valves may be directly related to ( J) glycerin stabilization and preservation of the collagen fibers, (2) the viability of the fibroblasts and macrophages within the implanted valves. and (3) the unique morphology and fine structure of the double-layered dura mater encephali . We hypothesize that the fibroblasts or myofibroblast-like cells found within the implanted leaflets. no matter what their origin. are capable of giving form and organization to the early developing connective tissue.
Delmas J. Allen, Ph.D., Toledo, Ohio, Gregory J. Highison, Ph.D.,** Reno, Nevada, Liberato 1. A. DiDio, M.D., Ph.D.,* Toledo, Ohio, E. J. Zerbini, M.D.,*** and L. B. Puig, M.D.,*** Sao Paulo, Brazil
Since the first implantation of biological valve prostheses in man, )-3 various types of tissue, including autologous and homologous fascia lata, heterologous pericardium, porcine heterografts, and homologous dura mater, have been used to replace defective human cardiac valves. As these valvular devices were functionally silent, nonhemolytic, and nonthrombogenic, the initial results obtained with bioprosthetic cardiac valve implantations were encouraging, but the durability of these valves has been questioned. The durability or long-term resistance to mechanical wear of the early
Address for reprints: Dr. Delmas J. Allen, Department of Anatomy, Medical College of Ohio, C.S. 10008, Toledo, Ohio 43699.
bioprosthetic cardiac valves was considered to be a function of host fibroblast infiltration and deposition of new connective tissue fibers. 4-6 Carpentier and associates? refuted the existing hypothesis that viability of the cardiac bioprosthesis was essential to long-term durability by demonstrating that host cell ingrowth was often more harmful than beneficial to the implanted cardiac bioprosthesis. They observed very little new collagen formation or host fibroblast infiltration in the aortic heterografts. Instead, Carpentier and colleagues 7 found a predominant host inflammatory cell infiltration leading them to conclude that long-term durability was dependent upon (1) prevention of host inflammatory cell infiltration, (2) reduction in the antigenicity of the graft, and (3) stabilization of the collagen fiber matrix with intramolecular cross-linkages. These postulates have since been widely accepted and confirmed by the proved long-term durability of the glutaraldehyde-treated porcine heterograft. At the ultrastructural level, the nonviability of the glutaraldehyde-treated porcine heterograft was demonstrated by Ferrans and associates. 8, 9 After noticing that
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From the Departments of Anatomy, Medical College of Ohio and University of Nevada School of Medical Sciences, and the Heart Institute, University of sao Paulo, sao Paulo, Brazil. Supported by grants to Dr. D. J. Allen from the American Heart Association, Northwestern Ohio Chapter, Inc., and the National Institutes of Health (BRS Grant 5-S01-RR-05700-II). Received for publication Sept. II, 1981. Accepted for publication Nov. 24, 1981.
0022-5223/82/080267+ 15$01.50/0
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Table I. Case data Case No. I
2
3 4 5 6 7 8 9 10 II
12
Age (yr)
31 29 44 7 18 33 48 57 36 50 29 37
Sex
Time of implantation (mo)
Valve location
Cause for reoperation
F F M M F F M M M M M M
97 100 59 23 67 50 70 66 108 27 90 66
Mitral Mitral Aortic Mitral Mitral Aortic Aortic Aortic Mitral Aortic Aortic Aortic
Calcification Calcification Rupture Calcification Rupture Rupture Rupture Rupture Rupture Rupture Endocarditis Rupture
progressive breakdown of collagen occurred in heterografts implanted longer than 2 months, they concluded that such breakdown was the crucial factor in determining the long-term durability of the glutaraldehydetreated porcine bioprosthesis, a deterioration that had not been reported by previous gross anatomic and histologic examinations.!? A comparison between the findings by Ferrans and associates" on the glutaraldehydetreated porcine heterograft and our" observations on the glycerin-treated dura mater allografts revealed that both bioprosthetic valves have equivalent long-term durability records, and the clinical success of both valves has been attributed to specific preimplantation treatment. Postimplantation surface alterations observed in the study of glycerin-treated dura mater valves were similar in many aspects to those described by Ferrans' group" for the glutaraldehyde-treated porcine heterografts. Surface fibrin formation appeared to be a natural response of vascular prostheses in general. However, a comparison of the cell types found within the glycerin-treated dura mater allografts and glutaraldehyde-treated porcine heterografts showed significant differences. In direct contrast, macrophages and fibroblasts were found throughout the implanted dura mater allografts. The primary objective of this investigation is to present subcellular or ultrastructural changes in dura mater valvular leaflets of a remodeling process which appears to be unique to this particular valvular prosthesis. Material and method
The twelve dura mater cardiac valves examined in this study were constructed, implanted into patients, and recovered at operation by Zerbini'" and his collaborators at the Heart Institute of the University of Sao
Paulo, Brazil. The list of the cases is given in Table I. The bioprostheses had to be removed because of valvular dysfunction. Dura mater encephali was removed from cadavers of 10- to 50-year-old human donors within 20 hours after death. Subjects with infective or degenerative diseases, as well as neoplasia, were excluded. After being rinsed in a continuous flow of water for I to 2 hours, the recovered dura mater encephali was placed in a sterile container of 98% glycerin at room temperature for 20 to 30 days before being constructed into valvular leaflets. 13 The aortic dura mater allografts recovered at operation after various periods of implantation (23 to 108 months) were gently irrigated with 0.8% heparinized physiological saline solution before placement in 3% glutaraldehyde solution (3 parts glutaraldehyde and 97 parts 0.144N sodium cacodylate buffer, pH 7.4). Upon arrival at the laboratory of the Department of Anatomy at the Medical College of Ohio at Toledo, the allografts were placed in the appropriate fresh fixation solution. Representative tissue samples were dissected from (I) the ring and strut areas of the support frame, (2) midline, and (3) along the free edge of both recovered and control valvular leaflets. Dura mater recovered at necropsy, preserved in 98% glycerin but not formed into bioprosthetic valves, and nonimplanted, glycerin-preserved dura mater valves were used as controls. Representative samples from each of the leaflets were rinsed in O.I44N sodium cacodylate buffer (pH 7.4) and processed for transmission electron microscopy by standard methods as described by Highison and associates. II In order to demonstrate connective tissue elements, sections were cut and stained according to the protocol described by Snodgrass." Stained ultrathin sections were examined and photographed with either a Hitachi llE-l electron microscope at 75 k V or a Phillips 300 electron microscope at 80 k V. Images were recorded on Kodak SO-163 film. Results
At the subcellular level, significant morphologic differences in the connective tissue fiber matrices and cell populations were noted when the recovered valvular leaflets were compared with the controls (cadaver dura mater and nonimplanted valves). Both types of control tissue were relatively acellular, and collagen (fibrils within each of the two layers (endosteal and meningeal) appeared to be arranged into parallel arrays or lamellae. In the recovered leaflets, however, distinct lamellae
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Dura mater cardiac valves
Fig. 1. Area of remodeling. Electron micrograph of portions of several fibroblasts (arrows) among the connective tissue components of recovered dura mater valvular leaflets. Intracellular and extracellular fine filaments are also present. The more densely stained orcein-positive material was always extracellular (arrowheads). Note the fine filamentous (wispy) material throughout the connective tissue matrix. (Orcein stain, xS,OOO.)
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Fig. 2. Remodeling in recovered valve. Portions of the nuclei and surrounding cytoplasm of at least two elongated fibroblasts predominate in this electron micrograph. Cytoplasmic and membrane-associated vesicles (arrow) are discernible at this magnification. Intracellular fine microfilaments and extracellular orcein-stained material are seen. Note the granular chromatin material with only a thin band lining the nuclear envelope. An occasional onionoid body (corpus cepiforme) can be visualized (arrowhead). (Orcein stain, x7,500.)
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Dura mater cardiac valves
Fig. 3. A fibroblast in a remodeling area. Numerous perinuclear small cytoplasmic vesicles measuring approximately 65 nm in diameter are present. Microfilaments and microtubules run parallel to the long axis of the cell subjacent to the cell membrane. Peripheral (marginal) filaments often form streamer-like processes (arrow) which extend from the cell surface suggesting motility. (Orcein stain, xS,OOO.)
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Fig. 4. Ultrastructure of cytoplasmic microfilaments and vesicles. Cytoplasmic vesicles (arrow) were observed in clusters among the cytoplasmic microfilaments. The beaded nature of the orcein-positive material and the relationship to the fibroblast are apparent at this magnification . (Orcein stain, x 15,380.)
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were usually absent after 23 months of implantation; these leaflets were composed of numerous cells and cellular processes with collagen fibers interspersed among them (Figs. 1 to 3). The collagen fibrils were arranged in criss-crossing bundles, as was evident from longitudinal, transverse, and tangential sections of collagen noted in the same electron microscopic field (Fig. 1). In low-power electron microscopic fields, both orcein-positive structures and areas of reabsorption were observed. The fibroblasts within valves implanted for 23 months and longer possess dense areas in and around their periphery (Figs. 2 to 4). At low magnifications (Figs. 2 and 3), these areas of peripheral condensation appear either as smudges (less dense) near the surface of the cell (Fig. 2) or as dark-staining beads (Fig. 4). The peripheral condensations observed in these cells (Figs. 2 to 4) also reveal a less dense, fibrillar component that is contiguous with the endoplasm. A parallel arrangement of delicate, threadlike or filamentous material that comprises the "endoplasmic condensation" is discernible (Fig. 4). These filaments show no evidence of periodicity or striations. The cell boundaries of these fibroblasts are not limited or marked by a distinct plasmalemma. This loss of a definitive outer cell membrane appears to be a distinguishing morphologic characteristic of cells undergoing peripheral and endoplasmic condensation. Fibroblasts found within these "rebuilding" areas possessed small cytoplasmic vesicles (Figs. 3 and 4) measuring 65 nm in diameter; these often were located in a peripheral position, suggesting that they were in the process of being extruded from the cell. Remodeling and turnover of connective tissue involve both the formation and degradation of collagen. The best evidence of early collagen formation in dura mater valves is found in association with peripheral filaments and peripheral condensation. Collagen fibrils, usually only a few bands in length, are found beside and seemingly within the peripheral filaments and condensations (Fig. 4). These short fibrils are usually amorphous, but they sometimes appear beaded. In accordance with present knowledge of connective tissue synthesis, the presence of the amorphous material with this positional relationship suggests that it contains precursors of collagen. Peripheral filaments may also form streamer-like extensions (Fig. 3) which project or trail from the fibroblasts, suggesting motility. In light microscopy, similar processes have been observed and referred to as stress fibers, tonofibrils, and fibroglia. Additional evidence for the formation or synthesis of collagen was found within the connective tissue matrices surround-
ing the cellular elements where electron-dense amorphous material was observed (Fig. 5). Finely granular and beaded fibrils with a cross-banding periodicity of 63 to 65 nm (the standard periodicity of collagen) between beads were located at the periphery of the amorphous component. Areas within the valvular leaflets where active degradation was adjudged to be occurring were characterized by the presence of lipid infiltration (Fig. 6) and macrophages (Figs. 7 to 9). Within these areas of remodeling and turnover of connective tissue fiber components, foci of progressive degradation were observed and characterized by (I) a decrease in the density of the collagen matrix, presenting with cleared-out areas (Fig. 7) and macrophages, (2) an increase in the extracellular fine filaments and granular material (Figs. 7 and 8), (3) lipid deposition, and (4) variability in the staining properties of the connective tissue ground substance and fibers. Macrophages were often observed within "cleared-out" or electron-lucent areas of the leaflets, believed to be caused by their phagocytosis and degradation of collagen (Fig. 7). These electron-lucent areas were usually surrounded by erratically staining connective tissue and ground substance. The macrophages possessed prominent multilobulated nuclei with heterochromatin located at the periphery of the nuclear membrane, few mitochondria and lysosomes, numerous clear and dense membrane-bound vesicles, and onionoid bodies in different stages of development or dense myelin-like figures." These phagocytic cells appeared singly and in small groups, but most frequently in large groups constituting nests (Fig. 9).
Discussion Since these were the only valves that needed surgical reimplantation, these studies are restricted so far to dura mater valves implanted for a minimum of 23 months. However, the data already obtained provide evidence of remodeling and turnover of connective tissue as revealed by active synthesis and degradation of collagen. Foremost among the observations reported herein are (1) that collagen synthesis appears to be related to certain morphologic characteristics of closely associated fibroblasts and (2) that collagen degradation occurs in or near areas where numerous active phagocytic cells were observed. The presence of either peripheral condensation or peripheral filaments in close proximity to both fibroblasts (regarded to be actively producing collagen) and mature collagen fibers was interpreted as evidence for collagen synthesis. In addition, the unique staining characteristics and special
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Fig. 5. Orcein-positive area within the connective tissue matrix. These orcein-positive areas within recovered valvular leaflets commonly demonstrated a centrally positioned amorphous component surrounded by fine granular and "beaded" material. The darker beads occurred at intervals averaging 65 nm. Thin sections treated with silver stain produced identical staining properties to those stained with orcein. (x 13,200.)
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Fig. 6. Lipid infiltration in recovered valve. A higher magnification of a selected area within a remodeling area revealed fenestrated or reticulated patterns (lipid droplets) and foci of variability in affinity for the orcein stain (darker areas). The connective tissue fibers in these areas also demonstrated a wide range in size. (Orcein stain, x 10,000.)
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Fig. 7. Macrophage within a cleared-out portion of a remodeling area . Macrophages, with numerous small plasmalemmal extensions similar to the one shown here , were observed within electron-lucent (cleared-out) areas . They were frequently surrounded by unevenly stained connective tissue fibers and a densely stained ground substance . (Uranyl acetate/lead citrate stain, x 14,000 .)
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Fig. 8. Transmission electron microscopic study of two macrophages located within connective tissue matrix of a recovered leaflet. The multilobulated nucleus with peripherally located dark-staining chromatin and fine plasmalemmal extensions are predominant morphologic traits of these cells. No cleared-out (electron-lucent) area surrounds these macrophages . (Uranyl acetate/lead citrate stain, x 13,200.)
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Fig. 9. A nidus of macrophages within a recovered leaflet. All of these cells possessed a multilobulated nucleus, Iysosomes, and residual or dense bodies . Dense amorphous material (left , middle) frequently appeared among these intrinsic cells and other connective tissue components . Intracellular and extracellular onionoid bodies (corpora cepiformia) are seen in different stages of development (arrows) . ( X 14.600 .)
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affinity of the filaments, fibrils, ground substance, and amorphous components for lead citrate/uranyl acetate, silver, and orcein stains provide further support for the synthesis of connective tissue fibers. In general, elastic fibers readily stain with orcein, whereas reticular fibers stain with both orcein and ammoniacal silver. Since only negligible amounts of elastic and reticular fibers were observed in our light and electron microscopic studies of dura mater and recovered dura mater valves, the term "orcein positive" is utilized herein to refer to precursors of collagen and new collagen. The densely staining beaded strands found throughout areas of remodeling are interpreted as newly formed acid-mucopolysaccharide-covered collagen fibrils rather than elastic fibers. Control dura mater leaflets were relatively acellular when compared with those of implanted valves. In areas of remodeling, considered to be sites where collagen synthesis was taking place, both active and inactive fibroblasts were identified, whereas in foci demonstrating degradation of collagen fibers, cells with ultrastructural characteristics of active macrophages predominated. No evidence of a degradative role for the fibroblast in the remodeling and turnover of collagen in dura mater valves was noted, as reported in soft connective tissue by others. 16 Many questions have emerged as a result of the findings reported herein. I. Did the fibroblasts found within the remodeling areas originate from the surrounding cardiac tissues or from the circulating blood of the host, or were they of dural origin, having survived the preimplantation processing? 2. Are the peripheral condensation and peripheral filaments part of the exoplasm of the fibroblasts, or are they secretory products of these cells? 3. A similar question could be asked about the macrophages found within the implanted leaflets, including a possible interconversion between fibroblast and macrophage.lv !" 4. Is the remodeling observed within the implanted allografts part of a normal repair mechanism initiated by the continuous wear and tear on the leaflets, or is it a degeneration/repair sequence in response to a pathological stimulus? 5. How is degeneration related to preservation and/or viability of implanted tissue? It is evident from the electron microscopic findings reported herein that significant morphologic changes occurred within the connective tissue substance of long-term (23 to 108 months) implanted dura mater
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allografts when compared to the control tissue. The implanted dura mater leaflets are definitely not biologically inert, as previously assumed. Viable cells (macrophages and fibroblasts) observed within the 12 dural valves investigated in this study appear to be responsible for and in the process of conducting phagocytosis and synthesis (replacement) of the fiber matrix of the collagenous connective tissue. Thus the long-term durability of dura mater bioprosthetic cardiac valves may be directly related to (I) glycerin stabilization and/or preservation of the collagen fibers, (2) the viability of the fibroblasts and macrophages within the implanted valves, and (3) the unique morphology and fine structure of the double-layered dura mater encephali. At present, since we have not had dura mater valves implanted for less than 23 months available for study, we can only hypothesize that the fibroblasts found within the implanted leaflets, no matter what their origin, are capable of at least giving form and organization to the early developing collagen fibrils. The peripheral filaments may be related or correspond to the strands of mucinous material pulling off the surface of fibroblasts, as described by Porter and Pappas'" in the chick. They demonstrated that this "mucin" at the fibroblast surface was amorphous. In light of our observations and what is known about connective tissue formation, it seems reasonable to postulate that amorphous components associated with the fibroblasts within the dura mater valves possessed collagen precursors. Since this amorphous material was also observed to have a certain affinity for ammoniacal silver stain (which is known to stain reticular fibers and acid-mucopolysaccharides), the material may be interpreted as having some relationship to the argyrophilia of "immature" or "developing" collagen (reticulin fibrils). In fact, many argyrophilic fibers often appear near and seem to be associated with fibroblasts according to early light'"- 20 and transmission':' electron microscopic reports. However, as these':': 19.20 and our observations were limited to mateials fixed for either light or electron microscopy, the actual dynamics of collagen formation by fibroblasts and degradation by macrophages remain speculative. Evidence of remodeling (degeneration/synthesis of connective tissue fibers) in the dura mater valvular leaflets was observed in the connective tissue matrix of the dura mater tissue itself. Morphologic changes at the transmission electron microscopic level indicative ofthe remodeling process described herein were not seen either in the area of the' 'fibrous sheath" originating from the recipient or at the boundary of this sheath and the original dura mater tissue. The cells found within these
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areas where remodeling was observed possessed physicalor morphologic characteristics of either phagocytes (macrophages and polymorphonuclear leukocytes) or fibroblasts. These cellular elements were present in both control tissue and implanted dura mater valvular leaflets, but were more abundant in the latter. Although the fibroblasts or fibroblast-like cells found within both control and implanted leaflets showed some features intermediate between classical fibroblasts and smooth muscle cells (myocytes), they were not identified as myocytes primarily because (I) they appeared as single isolated cells, (2) they did not possess a well-developed lamellar (Golgi) complex, (3) they demonstrated the absence of a moderately developed granular endoplasmic reticulum, (4) they lacked numerous free ribosomes and consistent accumulations of glycogen particles typical of myocytes, and (5) there were no nerve fibers and terminals associated with these cells. Despite the extensive scanning and transmission electron microscopic studies on dura mater by Allen and Low, 21 Allen and DiDio,22 and Allen and associates;" cells with features typical of smooth muscle observed in other viscera" have not been reported in dura mater. In conclusion, the most important points derived from this investigation are (I) that glycerin-treated and preserved dura mater allografts are not biologically inert in the human circulation, (2) that dura mater valves implanted for significant periods of time show signs of collagen degeneration which are similar to those seen in other tissue valves, most notably the glutaraldehyde-treated heterograft valves, (3) that valvular cellularity appears to increase with time in the dura mater valves, unlike glutaraldehyde-treated valves, and (4) that the possibility exists that host cellular infiltrates into the dura mater valve may actively participate in the removal of destroyed collagen and in the production of significant amounts of new host collagen. REFERENCES Murray G: Homologous aortic-valve-segment transplants as surgical treatment for aortic and mitral insufficiency. Angiology 7:466-471, 1956 2 Beall AC, Morris GC Jr, Cooley DA, De Bakey ME: Homotransplantation of the aortic valve. J THoRAc CARDlOVASC SURG 42:497-506, 1961 3 Kerwin AS, Lenkei SC, Wilson DR: Aortic-valve homograft in the treatment of aortic insufficiency. Report of nine cases, with one followed for six years. N Engl J Med 266:852-857, 1962 4 Carpentier A: Utilisation d 'heterogreffes dans Ie traitement des lesions de I'appareil valvulaire aortique. Etude biologique et technique. Premiers resultats cliniques. These Med, Paris, 1966
Thoracic and Cardiovascular Surgery
5 Angell WW, Angell JD, Kosek JC: Clinical and experimental comparisons establishing the glutaraldehyde treated xenograft as the standard for tissue heart valve replacement. Tissue Heart Valves, Mllonescu, ed., London, 1979, Butterworth & Co., Ltd., pp 89-126 6 Angell WW, Buch WS, lben AB: Formalin preservation of porcine heterografts, Biological Tissue in Heart Valve Replacement, MI lonescu, DN Ross, RH Wooler, eds .. London, 1972, Butterworth & Co., Ltd., pp 543-552 7 Carpentier A, Lemaigre G, Robert L, Carpentier S, Dubost C: Biological factors affecting long-term results of valvular heterografts. J THORAC CARDIOVASC SURG 58:467-482, 1969 8 Ferrans VJ, Spray TL, Billingham ME, Roberts We: Structural changes in glutaraldehyde-treated porcine heterografts used as substitute cardiac valves. Transmission and scanning electron microscopic observations in 12 patients. Am J CardioI41:1159-1184, 1978 9 Ferrans VJ, Spray TL, Billingham ME, Roberts WC: Ultrastructure of Hancock porcine valvular heterografts. Pre- and post-implantation changes. Circulation 58:Suppl 1:10-18, 1978 10 Spray TL, Roberts WC: Structural changes in porcine xenografts used as substitute cardiac valves. Gross and histological observations in 51 glutaraldehyde preserved Hancock valves in 41 patients. Am J CardioI40:319-330. 1977 II Highison GJ, Allen OJ, DiDio LJA, Zerbini EJ, Puig LB: Ultrastructural morphology of dura mater aortic allografts after 44-73 months of implantation. J Submicros Cytol 12:165-187, 1980 12 Zerbini EJ: Results of replacement of cardiac valves by homologous dura mater valves. Chest 67:706-710, 1975 13 Zerbini EJ, Puig LB: The dura mater allograft valve. Tissue Heart Valves, M1 lonescu, ed., London, 1979, Butterworth & Co., Ltd., pp 253-30 I 14 Snodgrass MJ: Ultrastructural distinction between reticular and collagenous fibers with an ammoniacal silver stain. Anat Rec 187:191-205, 1977 15 DiDio LJA. The ubiquity of "myelin figures" as seen with electron microscopy. Acta Biomedica 43:341-406, 1972 16 Ten Cate AR, Deporter DA: The degradative role of the fibroblast in the remodelling and turnover of collagen in soft connective tissue. Anat Rec 182: 1-41, 1974 17 Kouri J, Ancheta 0: Transformation of macro phages into fibroblasts. Exp Cell Res 71: 168-176, 1972 18 Porter KR, Pappas GD: Collagen formation by fibroblasts of the chick embryo dermis. J Biophysic Biochem Cytol 5:153-166,1959 19 Maximow A: Development of argyrophile and collagen fibers in tissue cultures. Proc Soc Exp Bioi Med 25:439442, 1927 20 Wolbach SB: Controlled formation of collagen and reticulum. A study of the source of intercellular substance in recovery from experiment scorbutus. Am J Pathol 9:689-700, 1933
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21 Allen 01, Low FN: Scanning electron microscopy of the subarachnoid space in the dog. 1 Comp Neur 161:515540, 1975 22 Allen OJ, DiDio LJA: Scanning and transmission electron microscopy of the encephalic meninges in dogs. J Submicr Cytol 9: 1-22, 1977 23 Allen OJ, DiDio LJA, Highison GJ, Puig LB, Zerbini EJ:
The importance of scanning electron microscopy in surgery as seen by the study of recovered dural cardiac valve prostheses. Biomed Res 2: 253-264, 1981 24 Amenta F, Allen OJ, DiDio LJA, Motta P: A transmission electron microscopic study of smooth muscle cells in the ovary of rabbits, cats, rats and mice. J Submicr Cytol 11:39-51, 1979
Notice of correction
In the September, 1979, issue of the JOURNAL, an error was made in an article by D. Craig Miller and associates, entitled "Operative Treatment of Aortic Dissections: Experience With 125 Patients Over a Sixteen-Year Period" (78:365-382, 1979). In the original publication, the figure 33, which is enclosed in the box at the bottom of the tenth column of the table shown below, read 22. Retrospectively, the 22 referred to the number of deaths following medical treatment of acute type B aortic dissections and had not been converted into the percent (22/67 = 33%). Recent reference in other manuscripts has led to the discovery of the error and indicates the advisability of this belated correction. Table IX. Operative or hospital mortality and sample size in selected series since 1970 Acute Type B
Type A Surgical Series Maryland UCLA Yale PBBH* SUNY-Upstate THI-Houston:j: Alabama THI -Houstont Memphis THI-Houston:j: Virginia Average Present study
Chronic
Medical
Surgical
Medical
Surgical
Medical
Surgical
Medical
%
No·1 %
No·1 %
No·1 %
No·1 %
I
Reference
Year
No·1
%
No·1
%
No·1
%
No.
10 12 14 15 16 17 22 19 20 21 66
1971 1972 1974 1974 1975 1975 1976 1976 1976 1977 1979
10
60 89 45 23 100
5
100
50
80 78 50
19 6 2
21
17
88
22
24 8 51 26
24 21 63 18 73
50 50 42 75 0 25 36
8
15 9 2
10 2 12 8 I 49§ II
25
80
14
43
3
100
14
36
9
179 53
38 34
76
83
121 20
36 45
67
1979
9 9 22 3
17
Type B
Type A
12
67
10
40
0
9
0
32
13 25 15
15 8 7
0
4 42 10
0 17 40
5
0
14
14
6
50
18
28
74 29
18 14
15
20
72 23
25 22
23
22
Ot
100 67
@]
Legend: The data in the original reports have been reclassified when necessary to correspond with the Stanford type A and B classification criteria. *Peter Bent Brigham Hospital (>53% of 45 total cases were acute). tAli patients with acute type B dissections failing medical therapy crossed over to surgical therapy. :j:Texas Heart Institute; some patients in reference 21 are included in reference 19. §Six of 12 patients who died had retrograde extensions of their dissections in perioperative period.