Artificial Organs 12(4):328-336, Raven Press, Ltd., New York 6 1988 International Society for Artificial Organs

Animal Evaluation of a New Pericardial Bioprosthe tic Heart Valve

T. J. Spyt, J. Fisher, "J. Reid, tJ. D. Anderson, and D. J. Wheatley

Department of Cardiac Surgery, Royal Infirmary, *Department of Veterinary Surgery, University of Glusgow, and ?Department of Pathology, Royal Infrmary,Glasgow, U .K .

Abstract: Implantation in animals is an essential step in the evaluation of any new prosthetic heart valve before commencing clinical trials. A new three-leaflet pericardial bioprosthesis developed in Glasgow has been implanted in the mitral position in ten sheep and eight dogs. Eleven animals were electively killed after 3 months of observa- tion and explanted valves were in good condition. Hydro-

Since the first clinical use of artificial heart valves in 1960, there have been continuing developments in their design, reflecting their less than ideal func- tion and propensity for complications.

Before commencing clinical evaluation of a new design of valve, it is necessary to implant the valves in animals in order to look for any possible adverse features that could not be predicted from the labo- ratory, hydrodynamic, and fatigue tests that have been undertaken.

Prosthetic heart valves have been implanted in both the left and right heart in sheep ( l ) , dogs (2), calves (3), and goats (4). The best choice of animal model in which to evaluate a prosthetic heart valve, and the best site in the heart to test a new valve, remains controversial. Sheep have been widely used for studying calcification in bioprostheses. Calcification can occur within a few months in bio- prosthetic valves when implanted in young, grow- ing sheep (1,5-10). The incidence of calcification in bioprostheses implanted in mature sheep has not been reported. Although bioprostheses have been successfully implanted in dogs (2,9,11) it has been

Received November 1987; revised March 1988. Address correspondence and reprint requests to Mr. T. J.

Spyt, Senior Registrar, Department of Cardiac Surgery, Royal Infirmary, Glasgow G21 2ER, U.K.

dynamic tests of the explanted valves showed small changes in function compared to tests prior tc1 implanta- tion. This was mainly due to host tissue ingrowth over the edge of the leaflets. Histological studies con&med good preservation of the pericardial tissue in explanted valves. Key Words: Pericardial bioprosthesis-Animal evalua- tion.

suggested that they are prone to infection and thrombosis (9,12). Dogs usually require a smaller prosthetic heart valve than sheep. The heart in young calves of about 70 kg is similar in size to that of adult humans (3). However, the size of the calf can double during a 3 months implantation period, making the prosthesis relatively stenotic, and in- creasing the risk of paravalve leaks (13). Calcifica- tion can also occur very quickly in young calves (13,14). Valves have been implanted in both the mi- tral and tricuspid sites in all three animal models. The right ventricle does not produce the same pres- sures and stresses on the right atrioventricular valve as are obtained in the systemic: ventricle. Since high mechanical stresses can initlate tissue damage and calcification (15), the tricuspid site is not as demanding a test for the prosthesis as is the mitral site.

A new pericardial bioprosthetic heart valve has been designed and developed in our Department and has been evaluated in vitro in our laboratory. The valve has a unique frame design with the valve leaflets mounted on an array of radically projecting pins, which allows leaflets to be interchanged dur- ing valve manufacture. Leaflets on each valve are matched for synchronous function under pulsable flow conditions during valve assembly. The frame is covered with a single piece of chemically treated

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NEW HEART VALVE 329

TABLE 1. Haemodynamic measurements of cardiac output and maximum pressure difference across the

valve prior to killing

sults of this animal evaluation form the basis of this communication.

Maximum pressure difference during

Cardiac output diastole Animal number (L/min) (mm Hg)

Sheep 6 2.4 4 Sheep 7 2.2 3 Dog 15 2.6 7.5 (thrombus)

bovine pericardium. Laboratory tests have shown that the long-term durability of this new valve is superior to other pericardial valves, with the elim- ination of leaflet tears found in other valves, caused by abrasion of the leaflets at the edge of their cloth- covered frames (16). Prior to clinical use of this new bioprosthetic valve, implantation was undertaken in ten sheep and eight dogs. Our experience and re-

MATERIAL AND METHODS Valve implantation in sheep

The bioprosthesis was implanted in ten female Greyface sheep, aged 18-20 months, and weighing 45-60 kg. Each animal was deprived of food for 48 h, and water for 24 h, prior to operation. After pre- medication with diazepam, anaesthesia was in- duced with Methohexiton. A cuffed endotracheal tube was inserted with the animal supine and a stomach tube was placed in the dorsal sac of the rumen. Anaesthesia was maintained with 1-2% halothane in oxygen. The chest was entered through the fourth or fifth left intercostal space. Ex- tracorporeal circulation was established with ve- nous drainage through a cannula inserted via the pulmonary artery into the right ventricle and arte-

FIG. 1. Photograph of the outflow aspects of two explanted sheep valves (numbers 7 and 10). Valve 7 showed some cal- cification. Valve 10 was free from calcification.

FIG. 2. Radiograph ot two exptanreo sriaep vaivas (iiurrioers 7 and 10) showing some calcification in valve 7.

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330 T. J . SPYT ET AL.

rial return into the descending aorta. A left ventric- ular vent was used to ensure decompression of the heart. A roller pump was used with a bubble oxy- genator, cardiotomy reservoir, and arterial line fd- ter. The pump was primed with 2 L of Ringer’s lactate solution with the addition of bicarbonate, mannitol, and heparin. During normothermic car- diopulmonary bypass, flow rates of 3.5-4 L/min were achieved. Ventilation was discontinued and anaesthesia was maintained by halothane delivered through the gas inlet of the bubble oxygenator. The heart was electrically fibrillated to prevent air em- bolisation. The mitral valve was approached through an oblique incision from the tip of the ap- pendage to its base.

A 25 mm external diameter valve was inserted within the annulus, using interrupted mattress su- tures buttressed with Teflon pledgets (2-0 Tycron, Davis & Geck, Hampshire, U.K.) placed on the ventricular surface. The prosthesis was orientated so that a scalloped interstrut portion spanned the

left ventricular outflow tract. Postoperative pain was controlled by an intercostal nerve block before thoracotomy closure, and by the analgesics admin- istered intramuscularly. The animals were given a 5 day course of antibiotics. No anticoagulant or anti- platelet drugs were given at any time in the postop- erative period.

At 3 months (17), the animals were anaesthetised and the chest was opened through the previous tho- racotomy incision. Cardiac output was measured using the dye dilution technique. The pressure drop across the prosthesis was recorded by direct mea- surement of pressures in the left ventricle and the left atrium. The animal was then killed and a post- mortem examination was carried out with particular attention to the appearance of the prosthesis and adjacent endocardium and evidence of systemic embolism.

The hydrodynamic performance of each prosthe- sis used was measured in a pulsatile flow test appa- ratus (18) prior to implantation. These in vitro tests

FIG. 3. Photograph of the outflow aspect of two explanted dog valves (numbers 12 and 14) electively killed at 3 months.

FIG. 4. Radiograph of two explanted dog valves (numbers 12 and 14) both free from calcification.

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were repeated after implantation and the changes in forward flow pressure difference and regurgitant volumes were measured. Pulsatile flow tests were carried out at 60 beatslmin, a stroke volume of 60 ml, and a peak forward flow rate of 150 ml/s. This corresponds to a cardiac output of approximately 3 L/min. The mean pressure difference during for- ward flow and the regurgitant volumes per stroke were measured (18). Recordings of the valve leaflet dynamics were made on video tape. The valve was then preserved in 0.25% glutaraldehyde and a ra- diograph taken of the valve from the outflow sur- face to detect macroscopic calcification. The leaf- lets were then removed and a further radiograph was taken of individual leaflets. Valve leaflets were then examined histologically.

Valve implantation in dogs The bioprosthesis was also implanted in the mi-

tral site of eight dogs weighing 25-33 kg. The pro- cedure was similar to that described for the sheep. Twenty-one millimeter diameter valves were used.

RESULTS Two sheep died shortly after surgery from respi-

ratory failure. Their prosthetic valves were func- tioning normally. A third sheep developed pros- thetic endocarditis and died 3 weeks after surgery.

The remaining seven sheep were electively studied and killed at 13 or 14 weeks.

Two dogs died shortly after surgery. In a third dog, the valve had been implanted with a suture inadvertently looped around one of the posts of the valve. This animal died 3 weeks after surgery with a thrombosed prosthesis and cerebral embolism. A fourth dog developed prosthetic endocarditis and died 3 months after surgery. Its prosthesis was ob- structed by thrombus and vegetations. The remain- ing four dogs were electively studied and killed at 13 weeks.

Table 1 gives the results of the haemodynamic tests on two sheep and one dog electively studied after 13 weeks. The appearance of the valves at the time of killing was generally good but in two of the dogs, thrombus wa_s visible adherent to the inflow aspect of the commissures. This thrombus signifi- cantly interfered with valve function in one dog, and this animal also showed multiple embolic in- farcts in both kidneys. The valve thrombus grew Candida albicans on bacteriological culture. There was no thrombus adherent to any of the sheep im- plants.

All of the valves implanted in both sheep and dogs showed smooth host tissue ingrowth over the sewing ring and extending 1-2 mm onto the ventric- ular surface of the base of the valve leaflets. Tissue

TABLE 2. Summary of hydrodynamic test results before implant and after explant

Root mean Regurgitation square (RMS) Mean pressure

Animal Implant/ flow difference Closing Closed number explant (mu,) (mm Hg) (ml) (ml)

Sheep 2

Sheep 5

Sheep 6

Sheep 7

Sheep 8

Sheep 9

Sheep 10

Dog 12

Dog 13

Dog 14

Dog 15

I E I E I E I E I E I E I E

100 100 104 104 102 102 101 101 105 105 100 100 100 100 100 100 100

100 100 100 100

1.2 1.8 1.5 2.0 1.2 2.9 1.5 3.8 1.5 2.1 1.8 2.6 I .5 2.4 3.4 5.1 3.2

Not measured 3.5 5.5 2.8 5.0

4.8 5.4 4.8 4.2 4.3 6.7 5.3 4.1 5.0 4.8 5.0 7.0 5.1 4.4 2.3 2.8 2.5

2.8 2.0 2.8 1.9

2.9 2.8 2.5 1.7 2.1 1.2 2. I 0 2.7 1.4 2.0 1.9 2.4 1.6 2.3 0.1 2.0

1.4 0.2 2.4 1 .o

I = prior to animal implantation, E = following explantation.

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332 T. J . SPYT ET AL.

ingrowth did not extend onto the pericardial- covered inflow aspect of the prostheses. Figure 1 shows the outflow aspect of two explanted sheep valves and Fig. 2 shows the radiographic appear- ance of the same two valves. A thin layer of host tissue covered the cloth sewing ring and outer sleeve-covering and extended up to 1 to 2 mm over the outflow surface of the leaflets at the edge of the

frame. Early calcification was visible on the leaflets of four prostheses in sheep; none was seen on the dog implants. Calcification was seen at the commis- sures of valves 5 , 6 , and 7 and lower down the leaf- lets at the edge of the frame in valves 6 and 7. This was confirmed on the radiographic films, which also showed a small amount of calcification at the com- missure of one leaflet in valve 8. Valves 9 and 10

FIG. 5. Micrograph of pericardial tissue from an explanted sheep valve (a) and a control valve prior to implantation (b). The stain was haematoxylin and eosin; magnification was (a) ~270, (b) x340.

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112 lh extension ratio

FIG. 6. Result of uniaxial tensile tests on pericardial tissue before implantation and after explantation. The hatching shows the range of curves for tissue prior to implantation and after explantation.

were free of macroscopic calcification. No calcifi- cation was found on pericardial coverings of the valve frame. Tissue ingrowth did not occur over the pericardial covering on the inflow aspect of the frame.

Photographs of the outflow aspect of two valves implanted in dogs are shown in Fig. 3 and the ra- diographic appearances are shown in Fig. 4.

Results of the hydrodynamic tests on the valves before implantation and after explantation are given in Table 2. The mean pressure difference across the valve was increased after explantation. In all valves, the difference was significant (p < 0.01, paired t test). In valves 2, 5 , 6, and 7, this was caused by stiffening of the leaflets due to the calci- fication, while in the other sheep valves and dog valves, the tissue ingrowth over the edge of the leaf- lets restricted the full opening of the leaflets. The minimal pressure difference and flow required to open the valve leaflets was also greater in the ex- planted valves. Closing regurgitation was un- changed but the closed leakage was generally less as the tissue ingrowth sealed the cloth sewing ring.

Histological studies showed that the collagen structure of the pericardial tissue was well- preserved in the explanted valve leaflets (Fig. 5). The characteristic crimp of the collagen fibre bun- dles can be seen in both implanted and explanted

FIG. 7. Micrograph showing the presence of calcium in an explanted sheep valve leaflet. The stain was Von Kossa and rnagni- fication was ~€300.

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334 T. J . SPYT ET AL.

tissue. Mechanical tests showed that the tissue was significantly thickened and exhibited reduced ex- tensibility after explantation. Tissue thickness was 0.44 +- 0.04 mm before implantation and 0.50 & 0.03 mm after explantation. The difference was signs- cant (p < 0.01, Student t test). The results of uniax- ial tensile tests on 3 mm wide strips, cut circumfer- entially from the leaflets, are shown in Fig. 6 for

control valves before implantation, antd sheep valves after explantation. There was a signifcant reduction in the mean extension ratio at 1.8 N force after explantation from 15 * 4.2 compared to 22.9 & 4.95 in valves prior to implantation (p < 0.02, Stu- dent t test). This was probably due to permanent deformation of the relaxed length of the tissue after repeated cycling of the leaflets in vivo, and would

FIG. 8. Scanning electron micrograph of the inflow surface of a control valve prior to implantation (a) and the outflow surface of an explanted sheep valve (b) (magnification ~2400).

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correspond to the loss of some of the crimp in the fibrils.

Histological stains showed the presence of cal- cium in some of the explanted sheep valves (Fig. 7). Calcium was not found in the explanted dog valves. Scanning electron microscopy confirmed the histo- logical findings of good preservation of the collagen structure in a control valve prior to implantation and in the explanted valves (Fig. 8).

DISCUSSION

Implantation in animals is an essential step in the evaluation of any new prosthetic valve prior to ini- tial clinical evaluation (17). While detailed informa- tion about mechanical function and in vitro durabil- ity can be obtained in the laboratory, implantation in animals is the only way of studying the effect of biological processes such as calcification, thrombo- sis, and tissue ingrowth. No ideal animal model ex- ists and our studies are in accord with the work of others showing that sheep show valve calcification at accelerated rates (1,5-10) while dogs have a risk of endocarditis and thrombosis (12). This new peri- cardial valve has shown similar hydrodynamic func- tion but greatly improved durability in laboratory tests compared to other pericardial valves (16), and this study was carried out to test its short-term function in a biological environment prior to im- plantation in humans.

The explanted sheep valves showed no throm- bus. There were only small deposits of calcium and these had little effect on the hydrodynamic function of the explanted valves. The calcification appeared at various places on the valve leaflets although it tended to be close to the edge of the valve leaflet, where bending stresses were high. It has been sug- gested that high bending stresses cause calcification (15). No calcification was found on the pericardial covering of the valve frame, which is stationary and is not subject to cyclic loading or bending stresses. This is similar to the findings with polyurethane valves in calves, where the valve leaflets were heavily calcified, but stationary polyurethane mate- rial used to coat the valve frames did not calcify (19).

While the explanted dog valves were free from Calcification, one valve was thrombosed with asso- ciated infection, and small amounts of thrombus were found at the commissures of a second valve. This did not affect leaflet function in the hydrody- namic tests.

Hydrodynamic tests of the explanted valves showed changes in function compared to the same

valves tested prior to implantation. Host tissue in- growth on the outflow surface of the leaflet at the edge of the frame restricted the portion of the leaflet that could flex to the open position. This caused a significant increase in the measured pressure differ- ence after explantation. The thickening and reduc- tion in extensibility of the explanted leaflets proba- bly contributed to the increased pressure difference and increased minimal flow required to open the leaflets. The reduction in extensibility of the ex- planted tissue is probably due to a permanent de- formation of the unloaded leaflet and loss of some of the crimp in the collagen structure. The host tis- sue ingrowth sealed the sewing ring and reduced the closed valve regurgitation in the explanted valves. Histological examination showed good preservation of the collagen structure.

The animal implant studies have shown satisfac- tory short-term performance of this pericardial valve. This pericardial valve is now being used in clinical practice.

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REFERENCES Jones M, Barnhart GR, Chaves AM, et al. Experimental evaluation of bioprosthetic valves in sheep. In: Cohn LH, Galluci V, eds. Cardiac bioprosfheses. New York: Yorke Medical Books, 1982;275-92. Okamura K , Yuge I, Kawazoe H, Kitamura N, Imamura E, Konno S. A comparative study between glutaraldehyde pre- served aortic and pulmonary heterograft valves in dogs. J Cardiovasc Surg 1976;17: 178-86. Yarborough JN, Roberts WC, Reis RL. Structural alter- ations in tissue cardiac valves implanted in patients and in calves. J Thorac Cardiovasc Surg 1973;65:364-75. MacIver AG, Howarth WS. The fate of prosthetic leaflet heart valves implanted in animals. In: Williams D, ed. Bio- compatibility of implant materials. London: Sector Publish- ing, 1985:211-5. Barnhart GR, Ishihara T , Ferrans VJ, Jones M, McIntosh CL, Roberts WC. Intracuspal hematomas in bioprosthetic valves. Pathological findings and clinical implications. Cir- culation 1982;66(suppl 1): 167-71. Barnhart GR, Jones M, Ishihara T, Rose DM, Chavez AM, Ferrans VJ. Degeneration and calcification of bioprosthetic cardiac valves. Am J Pathot 1982;106:136-9. Barnhart GR, Jones M, Ishihara T , Chavez AM, Rose DM, Ferrans VJ. Failure of porcine aortic and bovine pericardial valves. An experimental model in young sheep. Circulation 1982;66(suppl 1): 150-3. Barnhart GR, Jones M, lshihara T, Chavez AM, Rose DM, Ferrans VJ. Bioprosthetic valve failure. J Thorac Cardio- vasc Surg 1982;83:618-31. Gabbay S, Bortolotti U, Cipolletti G, Wasserman F, Frater RWM. The Meadox unicusp pericardial bioprosthetic heart valve. Ann Thoruc Slug 1984;37:448-56. Jones M, Eidbo EE, Walters SM, Ferrans VJ, Clark RE. Effects of two types of pre-implantation processes on calci- fication of bioprosthetic valves. In: Yacoub M, Bodnar E , eds. Biological and bioprosthefic valves. New York: Yorke Medical Books, 1986:451-9. Reece IJ, Anderson JD, Wain WH, et al. A new porcine bioprosthesis: in vitro and in vivo evaluation. Life Support Syst 1985;3:207-27.

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336 T. J. SPYT ET AL.

12. Hancock JB, Forshaw PL, Kay MP. Gore-Tex in canine coronary artery bypass. J Thorac Cardiovasc Surg 1980;80:94-101.

13. Boncheck LI, Tatooles CJ, Braunwuld NS. Experimental cardiac surgery in the calf. Ann Thorac Surg 1967;3:211-7.

14. Black M, Drury PJ, Lawford PV, Wain WH. The design, development and animal testing of the Shefield-Wessex Bioprosthesis. Life Support Syst 1986;4: 127-9.

15. Deck JD, Thubrikar MJ, Nolan SP, Aouad J. Role of me- chanical stress in calcification of bioprostheses. In: Cohn LN, Galluci V , eds. Cardiac bioprosrheses. New York: Yorke Medical Books, 1982:293-305.

16. Fisher J, Wheatley DJ. An improved pencardial biopros- thetic heart valve, design and laboratory evaluation. Eur J Cardiovasc Surg 1987;1:71-9.

17. Draft International Standard IS0 5840, Cardiac Valve Pros- theses, October 1986.

18. Fisher J , Reece IJ, Wheatley DJ. In vitro evaluation of six mechanical and six bioprosthetic valves. Thoruc Cardiovasc Surg 1986;34:15742.

19. Herold M, Lo HB, Reul H, et al. The Helmholtz Institute tri-leaflet polyurethane heart valve prosthesis. Proceedings of the I1 International Conference Polyurethanes in Biomed- ical Engineering, Stuttgart, West Germany, 1986.

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