Ann Thorac Surg 2001;71:S375-S378
© 2001 The Society of Thoracic Surgeons
Basic research
Effect of altered hydration on the internal shear properties of porcine aortic valve cusps
Eric A. Talman, PhDa,
Derek R. Boughner, MD, PhDa
a The John P. Robarts Research Institute and Department of Medical Biophysics, University of Western Ontario, London, Ontario, Canada
Address reprint requests to Dr Boughner, Division of Cardiology, London Health Sciences Centre, University Campus, 339 Windemere Rd, PO Box 5339, London, ON, Canada N6A 5A5
Presented at the VIII International Symposium on Cardiac Bioprostheses, Cancun, Mexico, Nov 3–5, 2000.
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Abstract
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Background. Dehydration of tissue due to glutaraldehyde fixation has been reported and was examined in this study of porcine aortic valve cusps. The effect of altered hydration on cusp internal shear properties was also examined.
Methods. Hydration level was assessed by wet mass measurement of cusps stored in solutions for times up to 1000 minutes. Solutions used in this study included Hanks solution, porcine blood, 0.5% glutaraldehyde, and several dextran solutions. Shear testing was performed on physiologically hydrated, superhydrated, and dehydrated cusps.
Results. There was very little difference between the physiologic and superhydrated leaflets; however, dehydration caused significant stiffening with increased hysteresis and stress relaxation.
Conclusions. Glutaraldehyde has been shown to increase shear stiffness of valve cusps. Tissue dehydration also increased shear stiffness but increased stress relaxation and hysteresis, which was contrary to observations reported after glutaraldehyde fixation. The significant effect of dehydration on cusp mechanical properties does not account for the effects observed after glutaraldehyde fixation, but it demonstrates that hydration level is an important factor that affects internal shear properties of valve cusps.
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Introduction
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Bioprosthetic heart valves made from glutaraldehyde fixed porcine aortic heart valves have better hemocompatibility and hemodynamics than mechanical heart valves. However, their long-term durability is unsatisfactory, as they frequently fail through structural deterioration after 10 to 15 years postimplantation. Valve failure is commonly due to tearing of the valve cusps, usually in conjunction with calcification; however, failure without significant calcification also occurs [1]. Mechanical stress is a factor, as the implant location with the highest transvalvular stress, the mitral position, fails with the highest frequency [2], and tearing and calcification are commonly observed in areas of high tensile and bending stresses [3]. Therefore, cusp mechanical properties are important for valve function, and altering them can contribute to the failure of porcine valve bioprostheses.
Glutaraldehyde induces collagen cross-linking, thereby reducing antigenicity and increasing tissue stability [4]. Although it sterilizes the tissue and acts as an excellent preservative/storage solution, it also affects the mechanical properties of the tissue. The tensile properties of glutaraldehyde fixed porcine cusps are satisfactory [5], but reduced tissue impairs the complex bending deformation required during valve opening and closing [6]. Internal shear stiffness is increased more than 50-fold after glutaraldehyde fixation [7, 8], a change that may contribute to tissue disruption and calcification.
In addition, glutaraldehyde causes tissue dehydration [9, 10], which also may alter the mechanical properties. Uniaxial tensile testing of porcine valve cusps is dominated by stiff collagen fibers at higher stresses. The contribution of other structural components such as elastin and glycosaminoglycans to tissue properties, as well as the tensile behavior of the tissue at low stresses are not well defined but are important factors in valve cusp pliability. The simple alteration of cusp hydration will affect viscoelastic properties, altering the tissue stress during rapid opening and closing of the valve. The ability of structural fibers to move relative to one another is important for normal stress distribution within the cusp and alteration of tissue hydration will affect the internal loading of tissue. This in turn may contribute to degradation of the tissue and failure of the device.
The purpose of our study was to examine the effect of altered hydration on the internal shear properties of porcine aortic valve cusps.
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Material and methods
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The study consisted of two parts: (1) assessment of the effect of glutaraldehyde, plasma, and dextran solution on the mass of fresh valve cusps, and (2) evaluation of the shear properties of valve cusps with altered hydration levels.
Assessment of hydration level
Mass change of individual cusps was used to assess hydration changes. Porcine valves were excised within 2 hours of slaughter. The cusps were cut from the valves, blotted dry, and weighed using a 1-mg precision electronic balance. Each cusp was placed into the test solution. After 1, 2, 5, 10, 20, 60, 120, and 1000 minutes the cusps were removed and then blotted, weighed, and returned to the solution.
Treatments
The solutions used in this experiment included porcine blood, Hanks solution (physiologic saline), 0.5% phosphate buffered saline, and six solutions of dextran. Low–molecular weight dextran with an average molecular weight of 105 g/mole was dissolved in Hanks solution at concentrations of 400, 100 and 10 g/L. High–molecular weight dextran with an average molecular weight of 107 g/mole was dissolved at concentrations of 100, 10, and 1 g/L.
The treatments used to assess the effect the hydration level on the internal shear properties were chosen to span a wide range of levels to demonstrate extreme effects of hydration. The dehydrated cusps were stored in the high-concentration, low–molecular weight dextran solution for 20 minutes, the physiologic cusps in Hanks’ solution for 2 minutes, and the superhydrated cusps in Hanks’ solution for 40 minutes before testing. The sample sizes for the dehydrated, physiologic, and superhydrated test groups were 12, 11, and 14, respectively.
Shear testing
The method used for the shear testing was the same as described in our previous work, except that testing was performed in 37°C humidified air rather than in isotonic saline [7, 8]. Briefly, circular punch specimens 6 mm in diameter were glued between parallel plates and a shear deformation was applied using a piezo-electrical linear actuator while measuring the resulting force. After preconditioning, each sample was cycled between shear strains of up to 1.0 and –1.0 at a rate of 0.1 second-1. Figure 1 shows the averaged results of the shear testing. Each curve has four distinct sections that were fit, for analysis, to this equation:
Stress relaxation was measured for 100 seconds at a strain of 1.0, which was reached at a strain rate of 1.0 seconds-1. The stress relaxation was represented by the stress remaining at 100 seconds normalized by the initial stress.
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Fig 1. Shear stress versus shear strain results. The averaged shear stress versus strain response of the three treatment groups tested at a strain rate of 0.1. (Shaded boxes = dehydrated tissue; shaded circles = physiological tissue; open diamonds = superhydrated tissue.)
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Results
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Mass changes
Glutaraldehyde caused cusp mass to decrease by almost 20% after storage in Hanks solution but caused little mass change in freshly excised cusps (Fig 2). Glutaraldehyde fixation also eliminated the superhydration that Hanks solution caused in fresh cusps. Hanks solution appeared to cause greater tissue swelling than blood, although blood did cause swelling (Fig 3), suggesting that the tissue was dehydrated upon delivery to the laboratory. The effects of the various dextran solutions are also shown (Fig 3). The highest concentration solutions caused dehydration, whereas the lower concentrations caused swelling. There were reversals of these trends in four of the six dextran solutions after long duration.
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Fig 2. Relative mass change of cusps that were put into Hanks solution for 1000 minutes and transferred to 0.5% glutaraldehyde (•), or placed in glutaraldehyde and then transferred to Hanks solution ( ). Break in the time axis represents transfer of tissue between solutions. Error bars represent standard deviation and n = 18 for both groups.
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Fig 3. Relative mass change of cusps stored in eight solutions. Hanks solution (n = 12) and fresh blood (n = 12) are labeled. (LMWD = low-molecular weight dextran solution; HMWD = high–molecular weight dextran solution [n = 9 for dextran groups].) Numbers next to the dextran solution labels refer to the relative concentration of the solution (1 = lowest, 3 = highest).
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Shear properties
The mass change and the thickness of the shear test samples are shown in Table 1. The three groups of tissue spanned a wide range of hydration levels. Table 1 also illustrates the differences in tangent moduli at three strain levels for the treatment groups and shows the differences in the stress values at 0 strain for the treatment groups. The "negative" stress values at zero strain are a feature of the bidirectional loading protocol and were a consequence of the viscoelastic nature of the material. Table 1 also shows stress relaxation and hysteresis. Figure 1 shows the average shear stress versus strain relationships for the three groups of tissue tested at a strain rate of 0.1 second-1.
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Comment
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Using mass change to estimate changes in tissue hydration may not be as accurate as wet and dry mass measurement; however, it is nondestructive and permitted a time-series collection for each specimen. The high water content of the tissue and the solutions used in this study meant that most of the measured mass change was due to water movement. Lillie and colleagues [11] used a similar technique to alter the hydration level of purified elastin for dynamic mechanical testing, and determined that it changed tissue hydration in a controlled manner. The technique that we used to measure internal shear properties has been used previously [7, 8]. It differs trom traditional tensile testing in that it tests the three valve layers (ventricularis, spongiosa, and fibrosa) in series rather than in parallel, meaning that the response is dominated by the soft spongiosa rather than one of the stiffer layers.
The effect of glutaraldehyde on cusp mass (Fig 2) is likely to be mechanical rather than osmotic, as phosphate buffered glutaraldehyde has physiologic ion levels and no macromolecules. Glutaraldehyde likely shrinks the tissue, squeezing out water while immobilizing the collagen structure.
Fresh blood caused cusp mass to increase (Fig 3), but Hanks solution caused a greater mass increase, which brings into question the appropriateness of simple saline solution for valve tissue testing and storage. As expected, high-concentration dextran solutions caused mass to decrease, whereas low-concentration dextran solutions cause mass to increase. The low–molecular weight dextran apparently penetrated the tissue because the mass loss observed in the high concentration reversed. The mass increase observed in high–molecular weight dextran at the lower concentrations also reversed over time, suggesting that components such as elastin and glycosaminoglycans were being extracted from the tissue along with associated water.
The three groups of tissue used for the shear testing encompassed a wide range of tissue hydration. Cusp thickness correlated well with change in cusp mass (Table 1). The significant effect of tissue dehydration on cusp internal shear properties was apparent (Fig 1) and quantified in Table 1. The stiffness of the dehydrated tissue was significantly higher than the other two groups at 0 strain; but as the strain increased, the stiffness of the tissue groups become much closer. The viscous nature of the tissues, as shown by the greater hysteresis and stress relaxation, was increased by dehydrating the tissue but was unchanged by increased hydration.
The changes observed in cusp shear properties due to altered hydration were markedly different from those reported after glutaraldehyde fixation. The latter produced a much greater increase in stiffness and a significant reduction in hysteresis and stress relaxation [7]. It is likely that glutaraldehyde alters internal shear properties by increasing chemical cross-linking in the collagen network, whereas dehydration alters the mechanical behavior of the highly hydrated matrix material. Thickening this jelly-like material will alter the ability of the fibrous network to freely move within it, thereby causing stiffening and altering the viscous properties of the cusp.
The effect of dehydration in the absence of chemical cross-linking was examined with the dextran solutions. Not unexpectedly, the observed effects suggested that changes due to glutaraldehyde fixation are not caused by dehydration but more likely by chemical cross-linking. However, the significant effects observed highlight the potential importance of controlling tissue hydration in processing porcine valve tissue for the manufacture of bioprosthetic heart valves. When designing studies to support product development, it should be remembered that tissue properties are affected by the solutions used and that simple physiologic saline may not mimic blood very well. This may be especially important for valves manufactured using minimally cross-linked tissues, which are likely more susceptible to altered hydration than is glutaraldehyde-stabilized tissue.
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Acknowledgments
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This work was made possible by the financial support of the Heart and Stroke Foundation of Ontario.
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References
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Abstract
Introduction
