The Phvsical Properties of Bovine Pericardium: A Stud? of the Elffects of Stretching during Chemical Treatment in Glutaraldehyde

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1 The Phvsical Properties of Bovine Pericardium: A Stud? of the Elffects of Stretching during Chemical Treatment in Glutaraldehyde I. J. Reece, M.B., F.R.C.S., R. van Noort, B.Sc., D.Phil., T. R. P. Martin, B.Sc., M.Sc., D.Phil., and M. M. Black, M.Sc., Ph.D. ABSTRACT The uniaxial stresslstrain response of bovine pericardium up to a stress of 1 N/mm2 was studied. The tissue was chemically modified in O.s0/o buffered glutaraldehyde for 24 hours under various conditions of constraint. Four groups were studied: an unrestricted control group, a group held at their original length, a group stretched to a stress of 0.04 N/mm2, and a group stretched to a stress of 0.4 N/mm2 during treatment. Histological examination revealed changes in structural organization of the collagen, which explained the alterations in the stresslstrain response. A significant alteration in mechanical properties after glutaraldehyde treatment was observed, with considerable loss of extensibility for the prestretched tissues. A valve made from such a prestretched tissue may be stenotic and possibly subject to early failure. Clinical experience with tissue heart valves is increasing and is encouraging [l-31. Although the ideal cusp material has yet to be found, bovine pericardium has much to commend it and has the advantages of being plentiful and relatively inexpensive to obtain and process. In the evolving process of tissue valve design and manufacture, the use of an aldehyde has become standard practice. Glutaraldehyde is used to tan the cusp tissue of the Carpentier- Edwards, Hancock, and Ionescu-Shiley valves, and the valves are then stored in formaldehyde. Glutaraldehyde reduces the antigenic- From the Departments of Cardiothoracic Surgery and Medical Physics and Clinical Engineering, Royal Hallamshire Hospital, and the Department of Restorative Dentistry, Charles Clifford Dental Hospital, Sheffield, England. Accepted for publication July 16, Address reprint requests to Mr. Reece, Department of Cardiothoracic Surgery, Royal Hallamshire Hospital, Glossop RD, Sheffield S10 2JF, England. ity of bovine pericardium and suppresses bacterial growth during storage [4], but its effect on mechanical properties of tissue has received little attention [5, 61. Details of the treatment method, such as glutaraldehyde concentration, ph, temperature, and duration of treatment, as well as the thickness of the material, are all thought to be important in determining the final physical properties. However, no attention has been given to the effect of stretching during treatment. Bovine pericardium can be stretched easily during handling, and this study was designed to investigate the effect that uniaxial prestretching has on physical properties after chemical modification with glutaraldehyde. Material and Method Sample Collection and Specimen Preparation Bovine pericardium was collected from animals aged 18 to 24 months from the local abattoir. It was stripped and cleaned of all fatty tissue, and, using a special cutter, was cut into standardsized dumbbell shapes, as shown in Figure 1. Areas were selected that would be of comparable thickness on gross examination. The thickness of each specimen (fo.o1 mm) was measured using a Mitsutoyo thickness gauge, and fine black silk markers were glued to the visceral surface with cyanoacrylate cement. The distance between the markers was accurately measured (ko.01 mm) with a traveling microscope. This distance was used in subsequent calculation of strain and was termed the gauge length. The width also was noted. The specimens were then mounted in Perspex blocks using cyanoacrylate cement and were ready for testing. During the measuring sequence, great care was taken to ensure that the tissue was flaccid, since the gauge length of the unmounted by The Society of Thoracic Surgeons

2 481 Reece et al: Physical Properties of Bovine Pericardium Fig 1. Shape and dimensions of the pericardium specimens. specimen defined the zero stresslstrain state of the tissue. This was accomplished by floating the specimen in saline solution on a glass slide and then removing the solution by capillary action with absorbent paper. Experimental Procedure Experiments on the mounted specimens of bovine pericardium were performed on a specially designed tissue test rig, described elsewhere [7]. The principal features of the apparatus are as follows: The test specimens are fully immersed in 0.9% saline solution at a constant temperature. The linear displacement is produced by a printed servomotor and the force measured with a Statham load cell. The extension of the tissue between the markers is measured with a purpose-built TV extensometer. Feedback facilities are incorporated into the system for either load or displacement cycling between preset limits. A nondestructive cyclic test procedure was adopted. The load necessary for the specimen to reach a stress of 1 Nlmm2 was determined from the cross-sectional area of the specimen. The specimen was mounted in the tissue test rig operating at a temperature of 30 C and the grip separation adjusted in such a way that the gauge length as measured by the TV exten- someter was the same as measured previously with the traveling microscope. A single loadextension cycle was then performed at a rate of 10 mm per minute resulting in a strain rate of approximately 50% per minute. The resultant forcelextension curve was plotted on an XY recorder. The force extension curves were then digitized using a Tektronix graphics tablet, interfaced with a Data General Nova Computer. By means of the appropriate cross-sectional area and gauge length, the stresslstrain relationships for each of the specimens was calculated and plotted on a Hewlett-Packard fourcolor plotter. After this initial procedure, each specimen was immersed in 98% glycerol to remove the tissue disturbance introduced by the tensile test. The success of the experimental procedure is dependent on the ability of glycerol to remove the history of any previous experiment performed on the tissue. This was evaluated on 3 specimens stored in glycerol. Each specimen was stored for one, four, and seven days at room temperature, and tested after each period had elapsed. Prior to testing, the specimens were reconstituted in 0.9% saline solution for a minimum of 1 hour. The resultant mechanical behavior for each of the stored tissues was compared with the stresslstrain response of the same tissue as fresh. A typical example of the results is shown in Figure 2. The repeatability of the experiment for the specimens tested was found to be within 3% variation, which was comparable to the accuracy of the test procedure. In all specimens there was complete recovery after storage in glycerol for 24 hours, and any permanent set introduced by the test was totally removed. Hence, the procedure was considered to be valid and the specimen could thus be used as its own control to assess the effects of further treatments [8]. Treatments A number of specimens were tested fresh as just described, and from these, four groups of 5 specimens each were selected. Selection was necessary in order to obtain four homogeneous groups in which the stresslstrain characteristics were similar. These specimens were placed in

3 482 The Annals of Thoracic Surgery Vol33 No 5 May N c m Fresh Day Days Days m E E m E 0.6- z m L 0) /*',/' #' Fresh - Fixed * '30 40 Strain in percent Fig 3. A typical stresslstrain relationship obtained for fresh pericardium illustrates the two areas of interest at a stress of 0.04 Nlmm2and 0.4 Nlmm2, which were used to define the strains for Groups 3 and 4, respectively. glycerol to remove the history of the nondestructive tests performed. The specimens were subsequently reconstituted from glycerol by immersion in 0.9% saline solution for no less than an hour. Each group of 5 specimens was then treated as follows: Group 1. These specimens were laid flat and unrestricted during fixation in 0.5% buffered Shiley glutaraldehyde (ph 7.4) for 24 hours at 22" f 1 C. This group served as the glutaraldehyde-treated controls. Group 2. The same treatment was employed as in Group 1, but these specimens were held at their gauge length on a special jig during fixation. Group 3. These specimens were stressed to 0.04 N/mm2 in the jig and held during fixation as described. Group 4. These specimens were stressed to 0.04 N/mm2 in the jig and held during fixation as described. The strains at stresses of 0.04 N/mm2 and 0.4 N/mm2 for Groups 3 and 4, respectively, were obtained by study of the stresslstrain relationships of the fresh tissue response. The levels of stress at 0.04 N/mm2 and 0.4 N/mm2 were chosen to coincide with the slow-rising early phase of the stresslstrain response and the later steep-rising phase as shown in Figure 3. These stresses represented an average strain of 21.7% for Group 3 and 29% for Group 4. After treatment in glutaraldehyde, each specimen was retested and the results compared with the corresponding fresh stresslstrain curve. Histology Transverse and longitudinal sections were cut from one specimen of each group after the second test following a further 24-hour period of glycerol treatment to remove the history of the test experiment, and were reconstituted with 0.9% saline solution. Light micrographs and electron micrographs were obtained for comparison of the groups. Results Mechanical Testing The specimens in Group 1 demonstrated increased stiffness at low levels of stress after chemical treatment, as shown in Figure 4. In the example shown, the fresh specimen can be stretched to 25% strain at 0.2 N/mm2 but to only 8% after glutaraldehyde treatment.

4 483 Reece et al: Physical Properties of Bovine Pericardium 1.2- ture of interwoven collagen bundles, which is Fresh also obvious from the electron micrograph shown in Figure 7. Histological study and electron microscopy of specimens from Groups 1 and 2 also exhibited this same wavy interwoven structure. However, specimens from Groups 3 and 4 were seen to have a dense central concentration of collagen (Fig 8) that, under the electron microscope, was seen to consist of 10,I densely packed parallel bundles of collagen fibers (Fig 9). Strain in percent Fig 5. Composite figure of a stresslstrain result selected from each of the four groups (1,2,3,4) illustrating the marked changes brought about by the application of stress during fixation. Group 2 specimens demonstrated similar changes. In Groups 3 and 4, the changes were more marked with very high stresses at low levels of strain. A composite of the different stresslstrain responses is shown in Figure 5 with a typical curve selected from each group, and illustrates the marked changes brought about by the application of stress during chemical modification. Histological Studies The histological appearance of fresh pericardium is shown in Figure 6. It has a wavy struc- Fig 6. Histological appearance of fresh pericardium. (van Gieson; xl00 before 15% reduction.) Comment Bovine pericardium is a thin and very flexible tissue that may be easily stretched during manipulation, as in manufacture of prosthetic heart valves. The results of our present study show that if the material is under tension during its period of chemical treatment, its me- chanical behavior in the direction of the uniaxial stress is significantly altered. The greater the applied stress, the stiffer the tissue becomes and the greater is the loss of flexibility. It has been shown by Cataloglu and associates [9] that human aortic cusps are subjected to stresses of 0.2 to 0.4 N/mm2; it is in just this physiological range that the greatest alteration occurs in the mechanical properties of bovine pericardium, especially when fixed under stress.

5 484 The Annals of Thoracic Surgery Vol33 No 5 May 1982 Fig 7. Transmission electron micrograph of fresh pericardium showing the wavy collagen pattern. (X12,600). Fig 8. A Group 3 specimen showing a diminished waviness and dense central collagen bundles. (van Gieson; xzoo before 15 reduction.)

6 485 Reece et al: Physical Properties of Bovine Pericardium Fig 9. Transmission electron micrograph of a Group 3 svecimen showing dense parallel collazen fibers. In Groups 3 and 4, the mechanical changes are consistent with the formation of dense central bands of uniformly oriented collagen bundles. The wavy structure is lost during stretching, and the glutaraldehyde treatment introduces collagen cross-linking, which maintains the stretched arrangement even when the deforming stress is removed. This accounts for the loss in extensibility. The implication of this result to the more complex biaxial loading situation in vivo is currently being investigated. Nonetheless it is clear that the need to avoid tissue stress during fixation must now be added to the list of precautions observed during manufacture of tissue valves. The simple stenting of a cusp with cotton wool during glutaraldehyde treatment may make the cusps more stiff than they would otherwise be. Such a valve would also be stenotic, as the cusp tissue lacks the relative flexibility of the glutaraldehyde-treated but unstressed tissue. Similarly, this loss of extensibility and flexibility of the prestressed tissue may cause higher stresses to be induced in the cusps during valve closure, and this, in turn, may promote early valve failure. References 1. ' Carpentier A, Deloche A, Relland J, et al: Six-year follow-up of glutaraldehyde-preserved heterografts. J Thorac Cardiovasc Surg 68:771, Ionescu MI, Tandon AP: The Ionescu-Shiley pericardial xenograft heart valve. In Ionescu MI (ed): Tissue Heart Valves. London, Butterworth, 1979, p Oyer PE, Miller DC, Stinson EB, et al: Clinical durability of the Hancock porcine bioprosthetic valve. J Thorac Cardiovasc Surg 80:824, Woodrooff EA: The chemistry and biology of aldehyde-treated tissue heart valve xenografts. In Ionescu MI (ed): Tissue Heart Valves. London, Butterworth, 1979, p Picha EJ, Gibbons DR: The effect of formaldehyde and glutaraldehyde on pericardium. In Williams DF (ed): Biocompatibility of Drugs and Materials. London, Sector Publishing, 1976, p Broom ND: Stress-strain behaviour of glutaraldehyde-preserved heart valve tissue. J Biomech 10:707, Van Noort R, Stevens JC, Black MM: A new apparatus for the in vitro testing of the mechanical properties of soft tissues. Engl Med 7:231, Van Noort R, Barker AT, Martin TRP, Black MM: The effect of glutaraldehyde and formaldehyde on the mechanical behaviour of bovine pericardium. In Transactions of the VII Annual Meeting of the American Society for Biomaterials. Troy, NY, May, 1981, p Cataloglu A, Clarke RE, Gould PL: Stress analysis of aortic valve leaflets with smoothed geometrical data. J Biomech 10:153, 1977