Pattern of arrangement of smooth muscle cells in neointimae of synthetic vascular prostheses

Pattern of arrangement of smooth muscle cells in neointimae of synthetic vascular prostheses Neointimae offabric vascular prostheses of three types (crimped and knitted [Aj, stretchable [Bj, and expansile [CV were examined under both light and electron microscopes from / to / ,240 days after implantation in the thoracic aorta of /6/ dogs. With all types of prostheses, a very uniform arrangement of smooth muscle cells was observed beneath the endothelial cells. In the crimped and knitted prostheses (AJ, the smooth muscle cells in the neointima showed a regular arrangement perpendicular to the direction of the bloodstream at each inner ridge of the crimp. In the stretchable prostheses (BJ, which can stretch only longitudinally, the long axes of the smooth muscle cells oriented in rows parallel to the bloodstream. In the expansile prostheses (CJ, which can expand only circumferentially, the smooth muscle cells were perpendicular to the bloodstream. These observations suggest that the arrangement of the smooth muscle cells in neointima is largely governed by the tension to which they are subjected.

Yasuharu Noishiki, M.D., Misasa, Japan

DUring the past three decades, many types of vascular grafts have been investigated in an attempt to find a useful replacement for diseased arterial segments.l" Among these grafts are synthetic fabric prostheses.v " Long-term studies have shown that the porosity of prostheses is important not only for proper healing of the prosthesis but also for prevention of regressive changes in the newly formed vascular wall. In the early experiments, no special attention was paid to elasticity of the prosthesis. However, experiments with elastic prostheses yielded satisfactory results.P" " and highly porous synthetic prostheses with some degree of elasticity were considered promising." In studies of the repair process of skin wounds, it has been ascertained that fibroblasts in granulation tissues are arranged in parallel rows and the direction of the rows is determined by the direction of the tension to which they are subjected. 14 The elasticity of a vascular prosthesis creates tension in the neointima during implantation. Thus we examined the relationship between the pattern of arrangement of new smooth muscle cells observed in the implanted vascular prostheses and the From the Institute for Thermal Spring Research, Department of Surgery, Misasa Branch Hospital, Okayama University, Misasa, Toltori 682-02, Japan. Supported in part by the Scientific Research Encouragement Grant from the Ministry of Education of Japan. Received for publication May 2, 1977. Accepted for publication Jan. 10, 1978.

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direction of the tension to which they are subjected. In studying the neointimae of fabric vascular prostheses of three types under both light and electron microscopes, we uncovered new evidence of the close relationship between cell arrangement and the direction of tension.

Materials and methods Synthetic vascular prostheses used. Three types of synthetic vascular prostheses were used in this study. Crimped and knitted prostheses (Type A) are marketed under the names of Cooley graft" and Weavenit." The other two kinds of crimpless prostheses are prepared from a special polyester cloth which can be stretched in only one direction: One of them (Type B) can stretch only longitudinally, and another one (Type C) can expand only circumferentially (Fig. I). In vivo experiments. As the test animals, 161 healthy mongrel dogs of both sexes weighing 8 to 12 kilograms were used. The left pleural cavity was entered through an incision in the sixth intercostal space with the dog under general endotracheal anesthesia. A 5.5 cm. segment of the thoracic aorta was resected and replaced by a prosthesis (5.7 em. long and 8 to 10 mm. in internal diameter). All anastomoses were performed with continuous suture of 5-0 Tevdek. Antibiotics were given at the time of operation, but no anticoagulant was used at any time. The specimens were removed from I to 1,240 days after the operations. Microscopic observation. The specimens were prepared in two ways for examination with the scanning

0022-5223/78/0675-0894$00.80/0 © 1978 The C. V. Mosby Co.

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r, j.

I

"

:

I,

,

" I

': i

I.

., I, 'I~ III;

I

Fig. 1. Preparation of the stretchable prosthesi s (Type B) and the expan sile pro sthesis (Type C). They are made of a special polyester cloth cut as shown in this figure.

Fig. 2. Schematic diagram of the apparatus for measuring the relative changes in length and circumference of the prostheses. See text under Materials and methods for explanat ion .

electron microscope: One group of specimens was fixed by 2 .5 percent glutaraldehyde in phosphate buffer (0.1 M , pH 7.4) and I percent osmium tetroxide at 4° c.; the other group was enzymatically dige sted by 0 .5 percent tryp sin in phosphate buffer (O.IM, pH 7.4, at 37° C . for 4 hours) before being fixed by glutaraldehyde and osmium tetroxide. The fixed specimens were deh ydrated in a graded series of ethanol and amyl acetate , critical-point dried with carbon dioxide;": 17 and coated with carbon and gold palladium. The studies were carried out with a JSM-50 A scanning electron

microscope (accelerating voltage 5 and 15 kv .) . The specimens for examination with the transmission electron microscope were dehydrated in a graded series of ethanol and propylene oxide and then embedded in Epon 812 . Ultrathin sections were cut on glas s knives wit h a JUM-7 ultratome and stained with uran yl acetate and lead citrate . I S These sections were examined and photographed with a JEM -100 B electron microscope (at 80 kv .) . In parallel , the specimens were examined under a light microscope after being fixed with a 10 percent solution of formalin.

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b

Fig. 3. Photomicrograph (a) and electron micrograph (b) of a crimped and knitted prosthesis (Type A) removed 573 days after implantation. a, The prosthesis is surrounded by connective tissue (hematoxylin and eosin; orig. mag. x 20). b, The surface of the prosthesis is covered with a continuous layer of endothelial cells (Ed). The subjacent loose collagenous tissue is occupied by elongated spindle-shaped cells. The bar indicates I /.I. (orig. mag. X5,OOO).

Fig. 4. Scanning electron micrograph of inner surface of neointima of a crimped and knitted prosthesis (Type A) removed 530 days after implantation . The arrow indicates the direction of the bloodstream. The bar indicates 10 /.I. .

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Fig. 5. High-power electron micrograph of inner surfaceof neointima of a crimpedand knitted prosthesis(Type A) removed 340 days afterimplantation. Beneath an endothelial cell (Ed), collagenfibers (Co), elasticfibers (Ef), and a smooth muscle cell (Sm) with basement membrane (Bm) are observed. The smooth muscle cell contains myofilaments (Mf), dense attachment (Da), and a pinocytotic vesicle (Pv). The bar indicates0.05 IJ-. (orig. mag. x 50,000). Measurement of tension. The direction of tension in the neointima caused by the internal pressure on each prosthesis was measured with the apparatus shown in Fig. 2. To both ends of a prosthesis (E), a solid rubber tube (B) and a rubber plug (F) were secured by ligatures. This set was dipped into a water bath (C), and water was conducted from a tap (A) through the rubber tube (B). The water pressure in the prosthesis was transmitted through a rubber tube (G) and converted into air pressure by a sealed bottle (l). Finally, the pressure was measured with a manometer (J). The prosthesis could be subjected to a desired pressure by changing the water pressure and could be expanded according to the pressure operating inside the prosthesis. The condition of the prosthesis at various degrees of inner pressure was photographed by a camera (K)

with a measure scale (D) through a sheet of glass (H) which was set on the surface of the water bath. The relative values of the changes in circumference and length of the prosthesis were calculated from the arithmetic mean value obtained from these photographs. The measurement of the relative changes indicates the direction of the tension in the neointima. Results In vivo experiments. Within 5 months after implantation, the synthetic prostheses were completely lined with a layer of tissue (Fig. 3, a). With all types of prostheses, the surface was covered with a continuous layer of endothelial cells with finely serrated and irregular borders. The long axis of the endothelial cells was parallel to the direction of the bloodstream (Fig. 4).

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Fig. 6. Scanning electron micrograph of the neointim a near one of the anastomotic lines of the crimped and knitted prosthesis (Type A) removed 526 days after implantation . This material is digested with trypsin to remove the endothelium before exam ination with the scanning electron microscope. The swelling at the lower left is the entering part of a suture thread . The bar indicates 50 IJ..

Beneath the sheet of the endothelial cells, a thin layer was observed. This layer was composed of long, slender cells. They are believed to be smooth muscle cells, because they have myofilaments, dense bodies, dense attachments , basement membranes , and pinocytotic vesicles, as shown in Fig. 5. Under the scanning electron microscope, the smooth muscle cells could be observed indirectly through the thin endothelial cells and looked like long swellings (Fig . 4). However, they could be examined directly when those endothelial cells were removed by enzymatic digestion." This was an effective means of observing the over-all pattern of arrangement of the smooth muscle cells (Figs . 6 and 7). In the crimped and knitted prostheses (Type A), a very uniform arrangement of the smooth muscle cells was observed at each inner ridge of crimps. The cells were positioned perpendicular to the bloodstream (Fig. 8) . On anastomotic lines , the smooth muscle cells near a stitch converged toward the stitch (Fig . 6) . In the

stretchable prostheses (Type B) and the expansile ones (Type C), the arrangement of the smooth muscle cells was very uniform also, but the direction of their arrangement was just the opposite; in Type B the long axis of the smooth muscle cells was parallel to the bloodstream , whereas in Type C it was perpendicular to the bloodstream (Fig. 7). Tension measurement. The relative changes in length and circumference, at both the wide; part and the narrow part of the vascular graft , were measured in all three types of prostheses . The changes in percent in relation to the inner pressure on the prosthesis (in millimeters of mercury) are shown in Fig. 9. In the crimped and knitted prostheses (Type A) , the inner ridge and the inner trench viewed from the lumen of the prosthesis were adopted as the narrow part and the wide part , respectively. The relative changes in the circumference at the inner ridge were greater than those at the inner trench, whereas the change was negligible along the length of the prosthesi s . In the stretchable pros-

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Synthetic vascular prostheses

Fig. 7. Scanning electron micrographs of tripsin-digested neointimae of (a) the stretchable prosthesis (Type B) removed at 406 days and (b) the expansile prosthesis (Type C) removed at 519 days. The arrows indicate the direction of the bloodstream. The bar indicates 40 f.t (orig. mag. x200).

Fig. 8. Low-power scanning electron micrograph of an inner surface of the neointima of a crimped and knitted prosthesis (Type A) removed 526 days after implantation. The inset shows relatively higher magnification of a part of an inner ridge. The arrow indicates the direction of the bloodstream. The bar indicates I mm.

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stretchable graft

crimped and knitted graft (WEAVENIT)

expansible graft

115

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110

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inner pressure

40 80 120 160 200 240 0

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40 80 120 160 200 240

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Fig. 9. Relative changes in circumference and length of threekinds of prostheses at various inner pressures. The percent of change is expressed in relation to that value obtained at a fixed inner pressure of 40 mm. Hg. theses (Type B), there was almost no change in circumference. By contrast, in the expansile grafts (Type C), the change was negligible along the length of the prosthesis (Fig. 9).

Discussion In the present paper, the orientation of the smooth muscle cells beneath the endothelial cells in neointimae of three types of fabric vascular prostheses was examined. The structural and functional adaptations of the smooth muscle cells to the mechanical and continuous stress caused by pulsation were clarified as follows: 1. With all three types of prostheses, a very uniform arrangement of smooth muscle cells was observed beneath the endothelial cells in the neointimae. 2. In the stretchable prostheses (Type B), which can stretch only longitudinally, the long axes of the smooth muscle cells were oriented in rows parallel to the bloodstream; in the crimped and knitted grafts (Type A) and the expansile ones (Type C), the direction of the smooth muscle cells was perpendicular to the bloodstream in the thoracic aorta. These data indicate that the pattern of arrangement of the smooth muscle cells in neointima is dependent not on the direction of the bloodstream but on the tension to which they are subjected. Type A prostheses, which are commercially available and used widely in clinics, are usually crimped by heat to give additional elasticity and to prevent kinking. 13 The crimped waves are stretched

by inner pressure, because Dacron fibers themselves are not elastic. Consequently, the modulus of elasticity is greatest at the inner ridge, where the crimped wave is set most strongly. The smooth muscle cells at the inner ridge of each crimp should be affected most intensely by the tension of pulsation along the circumferential direction. Reflecting these circumstances, the smooth muscle cells were perpendicular to the bloodstream, and this direction corresponded to the direction of the greatest tension. Furthermore, the converging pattern of smooth muscle cells near the suture thread was indicative of the pattern of tension in this area, because the prosthesis near the suture was somewhat puckered and the tension in this area was directed toward the thread. The opposite arrangement of the smooth muscle cells in the Type B and Type C grafts and the results of the tension measurements offered additional evidence in support of this conclusion. In studies of the repair process of skin wounds, it has already been noted that, in severed tendons," new fibroblasts infiltrating from neighboring tissues grow in parallel rows and the direction of these rows is determined by the tension to which they are subjected. Therefore, similar phenomena would be observed with other tissues which contain smooth muscle cells, e.g., organized tissue of mural thrombi": 22 or cellulofibrous intimal thickening of the aorta. 23 This type of cellular reaction against the blood pressure is considered to be one of the functional be-

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havioral patterns of the living body. The pattern of arrangement of the smooth muscle cells would make it possible to determine the prevailing direction of tension of the vascular prosthesis and, furthermore, the biological mechanism of adaptation of the living body to the prosthesis. The origin of the smooth muscle cells in question is still a matter of speculation. Haust and colleagues" examined the newly formed smooth muscle cells in the intimal lining of the injured surface of large vessels, and they suggested that the smooth muscle cells may be derived from the existing endothelium. However, Buck" reported that smooth muscle cells in the media multiplied and migrated through fenestrations in the intimal elastic lamina. In the case of neointima of synthetic vascular prostheses, mural thrombi that formed on the surface of the prostheses were shown to have been penetrated by cells migrating from contiguous parts of the vessel wall. In neointima located at some distance from the anastomotic ends, it seems likely that the cells from the anastomotic ends of the aorta do not migrate to the center of the prosthesis. Therefore, it is reasonable to assume that the endothelial cells turned into smooth muscle cells in situ. This point must be the subject of future research. I wish to thank Professor Y. Nakahara, Institute for Thermal Spring Research, Okayama University, and Professor T. Sunada, Department of Surgery II, Okayama University Medical School, for their valuable suggestions.

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REFERENCES Charles GR: A history of arterial surgery. Arch Surg 105:821-823, 1972 Kimoto S, Wada T, Yokota N, Hishida Y, Akiyama H: Late results of arterial grafts preserved in alcohol. Arch Surg 83:734-740, 1961 Creech 0 Jr, Deterling RA Jr, Edwards S, Julian OC, Linton RR, Shumacker H Jr: Vascular prostheses. Surgery 41:62-80, 1957 Voorhees AB Jr, Jaretzki A III, Blakemore AH: The use of tubes constructed from Vinyon "N" cloth in bridging arterial defects. Ann Surg 135:332-336, 1952 Wesolowski SA, Fries CC, McMahon JD, Martinez A: Evaluation of a new vascular prosthesis with optimal specifications. Surgery 59:40-56, 1966 Berger K, Sauvage LR, Rao AM, Wood SJ: Healing of arterial prostheses in man. Its incompleteness. Ann Surg 175:118-127, 1972 Yaw, PB, Grisell TW, Wahle WM, Shumacker HB, Glover JL: Fate of a nylon vascular prosthesis for aortic replacement. 14 year follow-up study. Surgery 75:140144, 1974

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8 Halpert B, De Bakey ME, Jordan GL Jr, Henly WS: The fate of homografts and prostheses of the human aorta. Surg Gynecol Obstet 111:659-674, 1960 9 Harrison JH, Davalos PA: Influence of porosity on synthetic grafts. Arch Surg 82:8-13, 1961 10 Sharp WV, Finelli AF, Falor WH, Ferraro JW Jr: Latex vascular prostheses. Patency rate and neointimization related to prosthesis lining and electrical conductivity. Circulation 29:Suppl I: 165-170, 1964 J I Ghidoni JJ, Liotta DD, Hall CW, Adams JG, Lachter A, Barrionueva M, O'Neal RM, De Bakey ME: Healing of pseudointimas in velour-lined, impermeable prostheses. Am J Pathol 53:375-389, 1968 12 Berkowitz HD, PerloffLJ, Roberts B: Pseudointimal development on microporous polyurethane lattices. Surgery 72:888-896, 1972 13 Lindenauer SM, Lavanway FM, Fry WF: Development of a velour vascular prosthesis, Current Topics in Surgical Research, vol 2, New York, 1970, Academic Press, Inc. 14 Anderson WAD: Pathology, ed 4, SI. Louis, 1961, The C. V. Mosby Company, p 56 15 Buxton BF, Wi.kasch DC, Martin C, Liebig WJ, Hallman GL, Cooley DA: Practical considerations in fabric vascular grafts. Introduction of a new bifurcated graft. Am J Surg 125:288-293, 1973 16 Anderson TF: Techniques for the preservation of threedimensional structure in preparing specimens for the electron microscope. Trans NY Acad Sci 13:130-134, 1951 17 Boyde A, Wood C: Preparation of animal tissue for surface-scanning electron microscopy. J Microsc 90:221 249, 1969 18 Frasca JM, Parks VR: Routine technique for doublestaining ultrathin sections using uranyl and lead salts. J Cell Bioi 25:157-161, 1965 19 Noishiki Y: SEM observation of smooth muscle cells in neointima of synthetic vascular prosthesis. J Electron Microsc (Tokyo) 26: 149-151, 1977 20 McMinn RMH, Pritchard JJ: Tissue Repair, vol I, New York, 1969, Academic Press, Inc. 21 Duguid JB: Thrombosis as a factor in the pathogenesis of coronary atherosclerosis. J Pathol 58:207-212, 1946 22 WoolfN, Robertson WB: An usual lesion in the pig aorta. J Pathol 103:231-238, 1971 23 Kojimahara M, Sekiya K, Ooneda G: Studies on the healing of arterial lesions in experimental hypertension. Virchows Arch [Pathol Anat] 354:150-160, 1971 24 Haust MD, More RH, Movat HZ: The role of smooth muscle cells in the fibrogenesis of atherosclerosis. Am J Pathol 37:377-389, 1960 25 Buck RC: Intimal thickening after ligature of arteries. An electron-microscopic study. Circ Res 9:418-426, 1961