Neointimas cultured in vitro for circulatory assistors

Neointimas cultured in vitro for circulatory assistors III Evaluation

0/ antithrombogenic properties

Minoru Adachi, M.D., Minoru Suzuki, M.D., lames N. Ross, lr., D. v.u.. Ph.D., David W. Wieting, Ph.D., lohn H. Kennedy, M.D., and Michael E. De Bakey, M.D., Houston, Texas

I

t has been established that when the inner surface of an implanted vascular prosthesis is covered with cell linings, the surface acquires anti thrombogenic properties. The living cell linings cultured in vitro have been used to cover the inner surface of circulatory assist devices and have been tested in experimental animals with some encouraging results.' The purpose of this study was to evaluate the anti thrombogenic properties of in vitro -cultured cell linings (neointimas) which covered the surface of supporting synthetic materials that were implanted as patch grafts in the right atria of dogs. Materials and methods

Forty-two adult mongrel dogs, weighing 20.5 to 33.0 kilograms, were divided into four groups. Prosthetic patches were implanted in the right atria of these animals. The right atrium of the heart was chosen From the Taub Laboratories for Mechanical Circulatory Support, Cora and Webb Mading Department of Surgery, and the Laboratory of Experimental Pathology, Department of Pathology, Baylor College of Medicine, Houston, Texas 77025. This study was supported by Research Grants HL-13330-03 and HL-05435-13 from the National Heart and Lung Institute, U. S. Public Health Service. Received for publication Feb. 14, 1973.

778

for evaluation of the anti thrombogenic properties of the materials in this study, because this chamber has the tendency to develop thrombus under low pressure and is technically suitable for implantation of the patch grafts without cardiopulmonary bypass. The following four types of patch grafts were used to evaluate the antithrombogenic properties of the inner surfaces: Group A-silicone rubber only; Group B-Dacron velour backed with silicone rubber; Group Cfibrin coagulum membrane (FCM) applied on the surface of Group B material; and Group D-autologous cell linings cultured in vitro on Group C material (Fig. 1). The preparation of FCM~ and the in vitro culture of cell linings on patch grafts used for the animals of Groups C and D have been described in detail.s:' Approximately 10' to 2 x 10' of omental cells prepared by trypsinization technique were inoculated on Dacron velour disc patches which had been coated with FCM in central areas. In order to ensure a selection of the best product, three patches with cell linings were prepared from each animal tissue. They were cultured in Eagle's medium with 10 per cent fetal calf serum in an atmosphere of 5 per cent carbon dioxide and 95 per cent air at 37° C. The in vitro incubation period ranged from

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GROUP A

L.-

GROUP B

l

GROUP C

--J1-

779

Silicone rubber

(QOOOQOQQOOOOOOOO- Dacron velour

J

Silicone rubber

ioooooooooOOOOOOg -~i: ~~nC~~~~~m L.

--.ll

membrane

Silicone rubber

GROUP D

Fig. 1. Four types of the patch grafts were implanted in the right atria of dogs.

3 to 7 days, averaging 5 days. For 3 dogs in Group D, autologous granulation tissue" was used for cell linings of the patches. The patches for Group C animals with FCM coating but without cell linings were incubated under the same condition used in Group D. At operation, a right thoracotomy was performed through the fourth intercostal space, and the pericardium over the right atrium was opened. A portion of the right atrium was occluded with a Satinsky partially occluding clamp and was incised longitudinally. One patch selected from the three prepared as above was trimmed aseptically into an elliptical form during the operation and was implanted in the right atrium of the animal. The dimension of the elliptical patches averaged 18 by 32 mm. The other two patches were used for preparation of histologic slides. The elliptical patch was sutured in the incised atrium with continuous 5-0 Ethiflex. The auricular appendage was avoided for the implantation. Air was evacuated before completion of the suture. No anticoagulant was administered locally or intravenously during or after the operation. Penicillin and streptomycin were administered for 5 days postoperatively. The dogs were housed in air-conditioned animal quarters and fed commercial canine chow ad libitum. They were killed by intravenous injection of sodium pentobarbital at 1 week, 2 weeks, 1 month, 3 months, and 6 months postoperatively (Table I) . At autopsy, the implanted patches were removed with the surrounding atrial tissue

Fig. 2. Silicone rubber grafts implanted in the right atria of dog hearts (Group A). A , One week. The inner surface of the prosthesis is covered with thick thrombus. B, Two weeks. A cross section of the patch graft shows silicone rubber at the bottom (arrows) and thick red thrombus covered wih fibrin at the top (magnified x2 ). C. Th ree months. The inner surface of the graft has organized thrombus with area s of calcification (arrow) .

from the hearts and were fixed in phosphatebuffered 10 per cent formalin. After fixation, the patch grafts were sectioned perpendicular to the surface , and the thickness of the inner layer, not including silicone rubber or Dacron velour of the prosthesis, was measured. Histologic sections were then obtained from the formalin-fixed patch grafts . They were embedded in paraffin, sectioned 7 p.

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Table I. Thickness (mean ± 1 standard deviation in millimeters) of inner layers of patch 7 Days Group: Type of patch

A:

Silicone rubber only B: Silicone rubber with Dacron velour

c:

Fibrin coagulum membrane on B D: Cells cultured in vitro on C

graft~

Postoperauw -. 30 Days

14 Days

Thickness

No. of dogs

3*

12.0 ± 1.3

2

12.5 ± 0.7

2

5.0 ± 4.2

2

3.3 ± 1.8

2

1.8 ± 0.4

2

4.3 ± 1.8

2

3.5 ± 2.1

2

3.8 ± 3.2

3

2.0 ± l.3t

2

2.3 ± OAt

4

1.8 ± l.8t

No. of dogs

I

I

Thickness

No. of dogs

I

Thickness 12.5

"One dog was killed on the second postoperative day. tSlllnlftcantly lower than the value of Group A (p < 0.01). XSlxteen dogs received patches with cultured autolo11aus omental cells. Three other dOllS received patch llrafta with cultun

thick, and stained with hematoxylin and eosin and Masson's trichrome methods. This experiment was conducted according to the principles enunciated in the Guide for Laboratory Animal Facilities and Care of the National Institutes of Health, United States Public Health Service. Results Table I shows the thickness of inner layers of four different types of patch grafts implanted in the right atria of dogs that were killed at various postoperative periods. Group A. Large thrombi were formed consistently on the silicone rubber patches of right atria of dogs killed from the second day to 3 months after the implantation. The thrombi were red initially but became white within I to 2 weeks (Fig. 2, A and B). The thickness of thrombi exceeded 10 mm. at any postoperative period. The organization of the atrial thrombus became evident from I to 3 month periods. There were areas of liquefaction and calcification in the thick thrombus (Fig. 2, C). Group B. Thrombi were also formed on the surface of Dacron velour bonded to silicone rubber which was used for this group. However, there was considerably less red thrombi than in Group A, ranging 1.5 to 8 mm. (Fig. 3, A and B). After 1 week, there were fibrin deposits without red

thrombi on the velour fabric of one patch. Three months after the implantation, the patch of 1 animal had become contracted, with organized tissue surrounding the velour (Fig. 3, C), whereas that from another animal had thin in vivo-formed cellular layers (neoendocardium), 2 mm. in thickness. Group C. Thrombus formation was noted on the surface of FCM, which had been applied on Dacron velour patches tested in this group. However, the development of red thrombus appeared less than that of Group A or B, and fibrin deposits were frequently noted on FCM (Fig. 4, A and B). The thickness of thrombi varied from 1.5 to 6 mm. In 2 animals the inner layers of the patches remained thin (1.5 and 2.5 mm.) after 1 month of implantation (Fig. 4, C).

Group D. Although thin thrombi were formed on some of the patches coated with precultured autologous cell linings which were tested in this group, the amount of red thrombus was small and the thickness of neoendocardium was significantly less than that found in Group A at any postoperative period (Table I). Deposits of thin fibrin were commonly noted directly on patches 1 week after implantation (Fig. 5, A). The surface of neointima appeared similar to that of Group C, but the amount of fibrin deposits in Group 0 appeared less than that

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implanted in the right atria of 42 dogs 1triod 90 Days No, of dogs

I

180 Days

Thickness

No. of dogs

I

Thickness

11.0

Total No. of dogs 7

Over-all mean thickness 12.1

±

1.0

2

3.8 ± 2.5

8

3.4 ± 2.3

2

4.3 ± 2.5

8

3.9 ± 1.9

6

1,5

19t

1.7 ± 1.2t

± 1.4t

4

1.3

± 0.3

lIllovlotion tissue.

in Group C. The fibrin deposits on the surfaces of Group C and D patches were not excessive. The fibrin deposits were not noted after 1 month, at which point the surface of the graft was smooth and glistening and lined with neoendocardium. The neoendocardia were usually thin enough to render Dacron velour visible (Fig. 5, B and C). The thickness of the neoendocardia in about half of the patch grafts in Group D dogs at 1 and 3 months measured 0.5 mm. or less (Fig. 5, D). The thickness of three patch grafts coated with autologous granulation tissue measured 2 mm. or less at 1 and 3 months, and their surfaces remained smooth (Fig. 5, E). Six months after implantation, the thickness of the neoendocardium had increased slightly, but the surfaces of the prostheses of 4 dogs remained smooth and glistening and were free of thrombi (Fig. 5, F). Microscopic features of the cell-lined patch grafts of Group D animals before and after implantation in the right atrium are shown in Fig. 6. Discussion

Silicone rubber has been used alone for some cardiovascular assistors. However, the results from Group A dogs tested with silicone rubber patches in this study were discouraging; the silicone had fewer antithrombogenic properties than did the other ma-

terials that were examined. Formation of thrombi was persistent and excessive over the silicone rubber prostheses in the right atria. In some of the Group B animals, the silicone rubber patches with bonded Dacron velour on the surface also developed thrombi, although not as severely and persistently as in Group A. In general, formation of thin fibrin deposits at the surface appears to be a crucial step for development of a smooth and thin inner layer over the prosthesis. In about half of the Group B dogs, the right atrial grafts had a smooth inner surface with a thickness of 2 mm. or less during the 3 month period. Thick red thrombi noted at 1 and 2 weeks on Dacron velour of Group B patches appear to have resulted in the thickened, irregular neoendocardia seen at I and 3 months. The patch grafts with in vitro coating of FCM, used in Group C, rendered the inner surface more compatible with the blood-prosthesis interaction than did those in Group A or B, since the fibrincoated surface was effective in preventing red thrombus formation during the immediate postoperative period. The FCM-coated surface of a cardiovascular prosthesis has induced good growth of smooth cellular linings in an in vitro experiment." The application of substrates, such as FCM and reconstituted collagen," may be useful in enhancing the growth of neoendocardium from sur-

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Fig. 3. Silicon rubber lined with Dacron velour (Group B). A, Two weeks. The inner surface of the prosthesis is covered by thrombus, with a central area of fibrin deposition. B, Two weeks. A cross section shows red thrombus over Dacron velour and fibrin deposit ion at the surface (arrows) (magnified x2 ). C, Three months. Organization of thrombus caused contraction of the graft.

Fig. 4. Fibrin coagulum membrane on surface (Group C). A, One week. The inner surface of the prosthesis is covered with fibrin. B, One month. The inner surface is smooth. C. Three months. A cross section of the prosthesis shows a smooth and thin inner layer (neoendocardium). A rrows point to the surface of neoendocardium (magnified x2 ).

rounding endocardium and in promoting the growth of implanted cells on circulatory assistors, e.g., atrial septal baffles and struts and seats of prosthetic valves. The inner layers of the cell-lined patches cultured in vitro and implanted in Group D dogs showed distinct antithrombogenic properties. They remained thin throughout the 6 month postoperative period. Their thickness decreased after 2 weeks, possibly as a result

of fibrinolysis of the initially formed fibrin deposits at the surface. Similar results were reported in neointirnas." Plasminogen in thrombus, in a nonocclusive form such as occurred in this experiment, can be converted to plasmin by an activator from circulating blood." ? The present experiment showed that fibrin was deposited on patches coated with autologous cell linings. However, an experiment by others revealed that

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Neointimas cultured for circulatory assistors

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Fig. S. Autologous cells explanted on surface and precuItured in vitro (Group D). A, One week. The inner surface of the prosthesis is covered with fibrin deposits. B, One month. The surface of the patch graft is smooth. Neoendocardium of this graft measured less than 0.5 mm. in thickness. C, Three months. Dacron velour is visible (arrow) through thin neoendocardium. D, Three months. A cross section of the graft shows thin and smooth neoendocardium. Arrows point to the surface (magnified x2). E, Six months. The neoendocardium is slightly thickened but its surface is smooth. F, Three months. A patch graft explanted with autologous granulation tissue, precuItured in vitro, and implanted to the right atrium. The inner surface is smooth.

the patches covered with autologous pericardium and jugular venous tissue caused formation of thrombi when implanted in the canine right atria." Thrombosis and fibrin deposition occurred on an autologous pericardial graft used as an atrial septal baffle.'! Fibrin deposition appears to be an important initial stage in the reconstruction of cardiovascular endothelium and in the maintenance of thin inner layers. The application

of living cell linings on prostheses should enhance the anti thrombogenic properties of the surface interaction of mechanical circulatory assistors.

Summary In order to evaluate the antithrombogenic properties of the inner layer of cardiovascular assistors, patch grafts with four different types of the surface were tested in the right

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Fig. 6. Photomicrographs of cell-lined patch grafts (Group D) . A , The inner layer of the prosthesis explanted with omental cells and cultured for 7 days in vitro . The surface has a smooth cellular lining. (Masson trichrome stain, x210.) B, En face view of omental cells explanted on prosthesis and cultured for 5 days in vitro. Abundant cellular growth is recognizable between Dacron velour threads. (May-Griinwald-Giemsa stain, x200.) C. Neoendocardium over the prosthesis 3 months after implantation in the right atrium. The neoendocardium has a superficial zone of loose parallel-lined cellular tissue and a deep zone of dense connective tissue. (Masson trichrome stain, x60. ) D, The deeper part of the neoendocardium in contact with Dacron velour of the graft 3 months after implantation. The interaction between neoendocardial tissue and Dacron threads is characterized by focal, foreign body, giant cell reaction. (Hematoxylin and eosin stain, x64.)

atria of dogs for up to 6 months postoperatively. Inner surfaces consisting of silicone rubber only, silicone rubber lined with Dacron velour, and Dacron velour coated with FCM were compared with Dacron velour surfaces coated with FCM and in vitro-cultured autologous cells. Thrombus formed most extensively on silicone rubber, consistently exceeding 10 mm. in thickness. The thrombi that formed on Dacron velour and on FCM-coated velour varied in thickness from 1.5 to 8 mm. There was no appreciable difference between these two groups. Thin inner layers formed in vivo over the prosthesis (neoendocardium), measuring 0.5 mm. or less, were observed only among the implanted patch grafts that had been lined with precultured autologous cells. Fibrino-

lytic action of the in vitro-cultured cellular lining (neointima) appears to contribute to the maintenance of thin neoendocardium. The application of living cell linings on cardiovascular prosthetic surfaces appears to minimize thrombus formation and prevent thickening of the neoendocardium. REFERENCES Bernhard, W. F., Husain, M., George, J. B., and Curtis, G -, W.: Fetal Fibroblasts as a Substratum for Pseudoendothelial Development on Prosthetic Surfaces, Surgery 66: 284, 1969. 2 Adachi, M., Suzuki, M., and Kennedy, J. H.: Preoperative Coating of Velour-Lined Circulatory Assist Devices With a Fibrin Coagulum Membrane (FCM), Trans. Am. Soc. Artif. Intern. Organs 16: 7, 1970. 3 Adachi, M., Suzuki, M., and Kennedy , J. H.:

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Neointimas Cultured In Vitro for Circulatory Assist Devices, L Comparison of Cultured Cells Derived From Autologous Tissues of Various Organs, J, Surg, Res. 11: 483, 1971. 4 Adachi, M., Suzuki, M., and Kennedy, J. H.: Neointimas Cultured In Vitro for Circulatory Assist Devices. II. A Light and Electron Microscopic Study on Cells Grown on Silicone Rubber Prosthesis, Cardiovas. Res. Cent. Bull. In press. 5 Adachi, M., Suzuki, M., and Kennedy, J. H.: Neointimas Cultured In Vitro for Circulatory Assist Devices. V. Use of Reconstituted Collagen as a Substrate. In preparation, 6 Warren, B. A., and Brock, L. G,: The Electron Microscopic Features and Fibrinolytic Properties of "Neo-Intima," Br. J. Exp. Pathol. 45: 612, 1964.

7 Fearnley, G. R.: Fibrinolysis by Adsorption, Nature 172: 544, 1953. 8 Von KauIla, K. N.: Chemistry of Thrombolysis, Springfield, III., 1963, Charles C Thomas, PUblisher, p. 229. 9 Jacobs, L. A., Klopp, E., and Gott, V. L.: Studies on the Finbrinolytic Removal of Thrombus From Prosthetic Surfaces, Trans. Am. Soc. Artif. Intern. Organs 14: 63, 1968, 10 Adachi, M" and Kennedy, J. H.: Unpublished data, 197 I. 11 Mohri, H" Barnes, R. W" Rittenhouse, E. A., Reichenbach, D. D., Dillard, D. H., and Merendino, K. A.: Fate of Autologous Pericardium and Dacron Fabric Used as Substitutes for Total Atrial Septum in Growing Animals, J. THORAC. CARDIOVASC. SURG. 59: 501, 1970,