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The role of elastin in aortic valve mechanics
Journal of Biomechanics 31 (1998) 115 — 123
The role of elastin in aortic valve mechanics Ivan Vesely Department of Biomedical Engineering, The Cleveland Clinic Foundation, Lerner Research Institute, 9500 Euclid Avenue, Cleveland, OH 44195, U.S.A. Received in final form 17 November 1997
Abstract Recent morphologic observations of elastin structures in aortic valves suggest that elastin is mechanically coupled to collagen. Since the mechanical stiffness of elastin is considerably lower than that of collagen, and aortic valves contain relatively little elastin, the mechanical importance of elastin in heart valve function is not clear. We have hypothesized that elastin acts to return the collagen fiber structure back to a resting configuration between loading cycles. The objectives of this research were therefore to elucidate the mechanical relationship between elastin and collagen structures within the aortic valve. To isolate elastin in a morphologically intact state, whole porcine aortic valve leaflets were digested in 0.1 N sodium hydroxide solution (NaOH) at a temperature of 75°C for 45 min. Elastin structures from the fibrosa and ventricularis were tested mechanically, and their loading curves compared to those of the original leaflet layers and to whole cusps. The elastin structures generated very low forces, having an elastic modulus only 0.05% that of the whole tissue. The contribution of elastin to tissue mechanics was significant at low strains and differed between the fibrosa and the ventricularis. Elastin tended to dominate the distensibility curves of the radial ventricularis, but participated very little in the fibrosa. The low but significant tensions produced by the elastin structures of the aortic valve, together with previously observed elastin morphology as well as the measurable preload of elastin, suggest that the purpose of elastin in the aortic valve leaflet is to maintain a specific collagen fiber configuration and return the fibers to this state, once external forces have been released. ( 1998 Elsevier Science Ltd. All rights reserved. Keywords: Elastin; Aortic valve; Collagen; Mechanics; Materials testing; Digestion
1. Introduction The aortic valve cusp is composed of two fibrous layers, the fibrosa and ventricularis, separated by a loose, gelatinous spongiosa (Gross and Kugel, 1931) (Fig. 1). These layers are very mobile and can easily compress and shear during leaflet flexure as the valve opens and closes (Vesely and Boughner, 1989; Song et al., 1990). The fibrosa and ventricularis are also preloaded by virtue of their attachment to each other; the fibrosa under compression and the ventricularis under tension (Vesely and Lozon, 1993; Vesely and Noseworthy, 1992; Vesely et al., 1993). It has been speculated that this preload results from the presence of elastin (Scott and Vesely, 1995). Unlike other highly elastic structures, such as the aorta, the valve cusps contain about 50% collagen and only 13% elastin by dry weight (Bashey et al., 1967). This
would suggest that, relative to collagen, the contribution of elastin to valve leaflet mechanics is minimal. However, during diastolic loading, there is considerable realignment of collagen fibers as the cusps extend beyond 50% strain and recoil elastically. Since collagen on its own is not highly elastic (extensible), we hypothesized that aortic valve elastin is responsible for their elastic recoil. This implies that collagen extends passively through most of its elongation phase. Indeed, recent morphological findings have suggested this mechanism (Scott and Vesely, 1996). The objective of this work was to measure the mechanical properties of aortic valve cusps and their elastin structures to elucidate the specific role of elastin in aortic valve leaflet mechanics, particularly in the recoil mechanism during unloading.
2. Methods * Corresponding author. Tel.: 001 216 445 6671; 216 444 9198; e-mail: [email protected]
fax:
001
0021-9290/98/$19.00 ( 1998 Elsevier Science Ltd. All rights reserved. PII S0021-9290(97)00122-X
The most direct way of measuring the function of elastin in connective tissues is to measure the mechanics
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I. Vesely / Journal of Biomechanics 31 (1998) 115—123
Fig. 1. A cutaway through the leaflet and aortic wall showing the internal configuration of the fibrosa, spongiosa and ventricularis. Near the central portion of the leaflet, the fibrosa exists as a folded sheet that unfolds as the leaflet stretches radially.
of whole specimens, remove all but the elastin, and then retest them. Roach and Burton (1957) used this approach to elucidate the relative contribution of collagen and elastin to the shape of the distensibility curve of arteries. Similarly, Oxlund and Viidik (1988) removed elastin and studied its effects on the biomechanics of skin. Autoclaving is perhaps the best technique for preparing intact elastin with unchanged mechanical properties (Lillie et al., 1994), but unfortunately it cannot be done simultaneously with mechanical testing. Based on amino acid analyses, Finlay and Steven (1973), Steven et al. (1974) and Hart et al. (1978) reported that NaOH and formic acid digestion are very good for purifying elastin. NaOH, in particular, produces elastin with intact inter-molecular cross links making it suitable for mechanical testing. Based on the work of Steven (1974), Song and Roach (1983) and Crissman (1987), we developed a technique to isolate morphologically intact aortic valve elastin. Aortic valve cusps were obtained fresh at the abattoir, typically from cross breeds of Duroc, Hampshire, Yorkshire, and Platrin pigs, usually slaughtered at 5—6 months, at a weight of 235—250 lb. These tissues were digested in 0.1 N NaOH at 75°C. The digestion time was optimized through time/digestion studies during which we examined thin sections stained with Gomori’s Trichrome. We also performed gravimetric studies and hydroxyproline assays, and determined that the optimal digestion time was 45 min. Under SEM, our tissue showed fibers, sheets, and open cells (Scott and Vesely, 1996) (Fig. 2a), structures similar to those reported by Song and Roach (1983), Yamazoe et al. (1990) and Crissman et al. (1987). We further verified the validity of these
Fig. 2. Typical SEM images of elastin structures isolated from aortic valves using NaOH digestion (Scott and Vesely, 1996). Note the wellpreserved fine detail of the elastin structures, particularly (a) a mesh of loose fibers coalescing into a sheet, and (b) an elastin tube exiting the leaflet near its attachment to the aortic wall.
structures by lyophilizing in water and in dimethyl sulfoxide (to prevent ice crystallization) and by critical point drying. In all three techniques, the morphology of elastin was similar. For mechanical testing, 1—3 leaflets from each valve were cut into 5 mm wide circumferential and 10 mm wide radial strips (Fig. 3) and tested in an Instron tensile testing machine (model 1125, Instron Corp., Canton, MA.), while immersed in a bath of Hanks solution heated to 37°C. The tissues were gripped in d320 emery paperlined aluminum grips and preconditioned by repeated stretching at 10 mm/min until reproducible load/elongation curves were attained. The preconditioning load was 5 g for circumferential and 10 g for radial strips. The plotted load/elongation curves were digitized on a Summagraphics tablet (model M11812, Fairfield, Conn.). Since the ‘noise band’ generated by the 100 g load cell was typically 50 mg, manual digitization in some cases smoothed the data. This produced a$0.2 mm uncertainty in gauge length.
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Fig. 3. Image of a typical aortic valve cusp, showing the region from which radial and circumferential test strips re cut. Test strips are always aligned along or transverse to the fibrosal ridges. Because of preconditioning effects, radial strips need to be 10 mm wide.
We took particular care in measuring the gauge length at loads as close to zero as possible. The tissue was clamped between the grips of the Instron and the grips moved together until the tissue was slightly wrinkled. The gain on the load amplifier was set to maximum and the grips slowly moved apart. As the wrinkles in the tissue straightened out, a slight increase in load was observed as the shaft with the grips was withdrawn from the water bath. Since the shaft was cylindrical, its buoyant force decreased linearly with extension until the tissue straightened and began to offer tensile resistance. At that point, the curve began to deviate from the linear plot produced by the buoyant effect. This was the point at which we chose the gauge length. Following testing in the fresh state, the tissue was slackened and the Hanks solution was replaced with preheated 0.1 N NaOH. A thermostatically controlled 1000 W heater maintained the temperature at 75°C during the 45 min of digestion. Following digestion, the digestate was drained, heated saline re-introduced and the digested tissue retested and fractured. Isolated fibrosa and ventricularis were subjected to the same tensile tests and digestions. By carefully grasping the ventricularis and pulling it away from the fibrosa with blunt forceps, the two layers could be separated along the mid-line of the spongiosa. Since it was not possible to measure the thickness of the specimens after digestion, stress could not be determined, and tension (defined as force per unit width of tissue) was used instead. It should also be emphasized that the concept of stress would be meaningless in highly porous structures, like the digested specimens, unless the area fraction of elastin is known. The main purpose of
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this study was to determine the relative mechanics of collagen and elastin, at very low loads. Such low-load experiments could be done under well-controlled conditions only under uniaxial loading. To analyze and compare the loading curves between tissues, these curves needed to be adjusted. We know from previous work (Vesely and Lozon, 1993; Vesely et al., 1993) that once separated from each other, the fibrosa elongates and the ventricularis shrinks, particularly in the radial direction. Once the ventricularis shrinks, its measured extensibility is greater than what it would be when part of the whole leaflet. This is because it was originally preloaded with its operating point slightly to the right of the origin of the extensibility curve. To duplicate this preload, its extensibility curve needs to be shifted to the left. Similarly, since the fibrosa unwrinkled and elongated, its apparent extensibility decreased, and the ‘wrinkled’ phase can be restored by shifting the curve to the right. Such shifting, however, must be done on loading curves where strain is expressed as natural strain, where e"ln [l/l ] (Vesely, 1996). 0 After each incremental shift of the fibrosa and ventricularis, these curves were arithmetically summed together and compared to the curve of the whole tissue. At each step, the ‘goodness-of-fit’ between the shifted and whole tissue curves was evaluated. The minimum of the error value between the whole tissue curve (obtained experimentally) and the layers’ curves (summation of fibrosa and ventricularis) was taken as the optimal shift interval. This shifting process is described in detail in (Vesely, 1996).
3. Results Isolated elastin was found to be extremely extensible, with a very low stiffness (Fig. 4). In the circumferential direction, the stiffness of aortic valve elastin is roughly 1/1700 of that of the whole cusp (2.1 N m~1 for elastin, vs 3.5 kN m~1 near max extension), and its extensibility is well over 100%. Its stiffness is also remarkably linear increasing from 0.31 N m~1 at low loads to 2.1 N/m~1 at high loads. This is consistent with our existing understanding of elastin structures, as reported by Fung (1981). The whole valve, on the other hand, increased its stiffness 1100 times (from 3.1 to 3500 N m~1 at high loads). We also found that elastin structures of the fibrosa and the ventricularis have very different mechanical behavior (Fig. 4). Based on the shape of the tension/strain curves and the low fracture strength, very little elastin exists within the fibrosa. The fracture tension of the fibrosal elastin was 0.35 and 0.14 N m~1 in the circumferential and radial directions, respectively, whereas elastin from the ventricularis had a mean fracture tension almost an order of magnitude greater, at 2.94 and 2.81 N m~1 (Table 1).
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Fig. 4. Original data curves for all the specimens tested in this study, converted to tension vs strain. These curves show the relative variability of the data, as well as the gross differences between the groups. The fresh tissues were stretched to arbitrary loads, and digested tissues were stretched to failure.
While elastin structures generate forces many times lower than collagen, they are not negligible. The relative contribution of elastin can be estimated through a comparison of their tension/strain curves, once these curves were appropriately shifted. The optimal shift values were e"0.25 and e"0.11 for the radial fibrosa and the ventricularis, respectively (Fig. 5). This means that the
fibrosa needed to be shifted by 0.25 to the right and the ventricularis by 0.11 to the left. Similarly, for circumferential tension curves, the optimal shift was at 0.11 for the fibrosa and 0.014 for the ventricularis (Fig. 5). Examining the shifted tension/strain curves, it can be seen that in the radial direction (Fig. 5d), the ventricularis appears to dominate the elastic response in the low-strain
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Fig. 5. Diagrammatic representation of the curve shifting process: (a) The original functions for the whole cusp, the fibrosa and the ventricularis, tested in the circumferential direction. Note that the fibrosa should be shifted to the right and the ventricularis to the left, before summing the curves together and comparing with the curve from the whole tissue. (b) Plots showing the shape of the circumferential fibrosa and ventricularis curves after optimal shifting. The fibrosa and the ventricularis were shifted by 0.25 and 0.11 strain units to the right and left, respectively. (c) The original functions for the whole cusp, the fibrosa and the ventricularis, tested in the radial direction. Note that the fibrosa should be shifted to the right and the ventricularis to the left before summing them together and comparing with the whole tissue. (d) Plots showing the shape of the radial fibrosa and ventricularis curves after optimal shifting. The fibrosa and the ventricularis were shifted by 0.11 and 0.014 strain units to the right and left, respectively.
region. At these low strains, the fibrosa does not contribute any tension since it is highly folded and is simply unfurling its coils. In the circumferential direction (Fig. 5b), a similar process occurs, but to a lesser extent. Below strains of 0.15, the fibrosa has little effect, between 0.15 and 0.25, the fibrosa and the ventricularis contribute almost equally, and above strains of 0.25 and loads of 2.0 N m~1, the fibrosa begins to dominate. Unlike the loading curves of the fibrosa and ventricularis that had to be reconstructed mathematically, the loading curves of elastin structures were adjusted
from direct measurements. Because the same tissue strip was tested before and after digestion without removal from the tissue grips its gauge length, and hence shrinkage, was measured directly. The isolated elastin structures shrank by 10—30% during digestion (Table 2), indicating that they were preloaded in tension when connected to collagen. Once the elastin curves were shifted by the appropriate amounts, it can be seen that in the fibrosa, the majority of the tension is generated by collagen (Fig. 6). This is because the elastin can generate only about 0.1 N m~1 of tension at physiological strains. In the ventricularis, however, the elastin structures
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dominate the mechanical response in the radial direction, while in the circumferential direction, the loads are shared almost equally between elastin and collagen up to 0.25 strain. The elastin plots of whole cusps are inconclusive, as different amounts of contraction likely took place in the elastin of the fibrosa and the ventricularis.
Table 1 Fracture parameters for elastin isolated from whole cusps and layers
Circumferential
Whole
Fibrosa
Ventricularis
Radial
Whole
Fibrosa
Ventricularis
N Mean S.E.M. N Mean S.E.M. N Mean S.E.M. N Mean S.E.M. N Mean S.E.M. N Mean S.E.M.
Fracture tension (N m~1)
Fracture strain(%)
15 2.72 0.29 7 0.35 0.06 8 2.94 0.29 8 1.65 0.36 10 0.14 0.01 10 2.81 0.30
15 143 10 7 101 12 8 152 12 8 127 17 10 147 13 10 170 12
4. Discussion In this study, we have characterized porcine aortic valve elastin and estimated its relative contribution to the mechanics of the fibrosa and ventricularis. Both the mechanical properties of elastin, as well as the changes in gauge length following digestion, confirm our hypothesis that elastin structures impose tensile forces on collagen fibers during valve unloading. In order to accept the results of this work, however, certain assumptions have to be made. First, we must assume that the mechanical behavior of tissues measured in vitro is representative of their behavior in vivo, under biaxial loading conditions. Experience has shown that while absolute values of elastic moduli and extensibility may differ from real, in vivo conditions, standardized uniaxial tensile tests, such as these, are extremely sensitive to differences in mechanics between test groups. Certainly, the poor aspect ratio of the radial tissue strips can be one source of error. The radial strips were chosen wider than the circumferential because strips of tissue narrower than 7 mm have been shown to fail during preconditioning (Lee et al., 1984). Although the low aspect ratio does not permit uniform uniaxial loading, these effects were present in both fresh and digested specimens, and would not likely affect the conclusions drawn regarding load sharing between elastin and collagen. The shrinkage of the digested tissues suggests denaturation. Mature elastin, however, is highly stable at elevated temperatures (Lillie et al., 1994) and is unlikely to be damaged, particularly since fine structures were
Table 2 Mean gauge length of fresh and digested aortic valve cusps and their components Difference
Circumferential
Whole
Fibrosa
Ventricularis
Whole
Radial
Fibrosa
Ventricularis
N Mean S.E.M. N Mean S.E.M. N Mean S.E.M. N Mean S.E.M. N Mean S.E.M. N Mean S.E.M.
Fresh (mm)
Digested (mm)
15 8.2 0.42 7 8.9 0.59 8 7.4 0.81 8 5.3 0.28 10 5.6 0.42 10 4.0 0.43
15 8.0 0.40 7 7.9 0.63 8 6.4 0.75 8 4.7 0.53 10 4.0 0.33 10 3.4 0.41
% strain
Natural strain
Significance
0.024
NS
11
0.10
p(0.088
14
0.13
p(0.0035
11
0.10
NS
29
0.25
p(0.00001
15
0.14
p(0.00001
2.4
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well preserved (Fig. 2). The longitudinal shrinkage therefore likely resulted from the release of some residual stress upon the removal of collagen, and is consistent with the observations of Oxlund et al. (1988).
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Perhaps the most important biomechanical effect of isolating elastin is that one removes the collagen fiber bundles that fill spaces within the elastin structures. Consequently, during stretching, the porous elastin structure
Fig. 6. Plots of the functions describing the mean behavior of fresh and digested tissues. Note the widely varying contribution of elastin to the function of the valve layers. However, while these plots can be used to determine the relative contribution of elastin to the behavior of the fibrosa and the ventricularis, it cannot be done for the whole tissue. The elastin curves for the whole tissue represent elastin from both the fibrosa and the ventricularis, which exist in some mixed form of preload. Since these elastin curves represent preloaded materials, they have not been shifted and therefore cannot be used for superposition.
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can collapse much more than it could have when the voids were filled with collagen. This likely increases the extensibility and compliance of the elastin. It is interesting to note, however, that the elastin of the radial ventricularis (Fig. 6) follows the original ventricularis curve very closely to almost 50% strain, suggesting that the elastin sheets of the ventricularis are flat, and do not have many voids or undulations that could collapse. This is consistent with our morphological observations (Scott and Vesely, 1996). Finally, the mechanical effects of the proteoglycans in the ground substance of the valve were not considered in this analysis. Proteoglycans bind intimately with collagen fibers and could significantly reinforce the collagen fiber matrix. The loads carried by ‘collagen’ should therefore be considered as a summation of loads distributed between collagen and proteoglycans. However, proteoglycans typically contribute mechanically only during dynamic loading and have minimal contribution statically. Indeed, Viidik et al. (1982), and more recently Kronick and Sacks (1994) have shown no significant effects of proteoglycan extraction on the static mechanical properties of biological tissues. In the context of the above assumptions, the results of this study offer an improved understanding of the internal micromechanics of aortic valve cusps and the functional role played by elastin structures. First of all, the mechanical data confirm the low proportion of elastin in heart valve leaflets (Bashey et al., 1967). Because of their porosity, these elastin structures have a maximal stiffness of only 10 kPa, more than an order of magnitude lower than the 400 kPa of the relatively pure elastin from ligamentum nuchae (Fung, 1981) (stiffness of valve elastin was estimated from Fig. 4, assuming a thickness of 0.5 mm). As the ventricularis is stretched in the radial direction, the dominant restorative force is the tension produced by elastin. This is not surprising, since the ventricularis must undergo extensions of up to 60% strain and collagen is relatively inextensible. The collagen is likely organized in a highly wavy configuration and uncoils passively through most of the extension of the ventricularis. Once extended fully, the collagen takes over the load and limits further extension. This was suggested by us previously (Vesely and Noseworthy, 1992) and has now been verified through direct mechanical testing. In the circumferential direction, however, collagen appears to play a more significant role, sharing load roughly equally with elastin up to the transition phase (&20% strain). In the fibrosa, elastin plays a minor role, both radially and circumferentially. In the radial direction, the fibrosa has a macroscopic waviness so that it can stretch to large strains passively until ‘lock-up’. Because of the extremely low mechanical contribution of fibrosal elastin (Fig. 6), the fibrosa likely relies on the ventricularis to pull it back into its folded shape.
The function of elastin is therefore highly dependant on its configuration and relative content in the given material. Unlike the low-modulus ‘elastin’ phase of the aorta (Roach and Burton, 1957), aortic valve elastin can have (i) a minimal contribution to mechanics, as in the fibrosa; (ii) can participate equally with collagen, as during initial stretching of the ventricularis circumferentially; and (iii) can also totally dominate the mechanics at the early phase, as in the radial ventricularis. It is important to note that elastin content alone does not dictate mechanics, but rather its organization relative to collagen. The ventricularis contains a considerable amount of sheet elastin (Scott and Vesely, 1996) with loading curves that appear identical radially and circumferentially (Fig. 4 and 6). Since the mechanics of the whole ventricularis are not identical in the radial and circumferential directions, it must be the amount of collagen or the types of connections between the elastin and collagen structures, that are responsible for this anisotropy, not the mechanics of the elastin itself. We have shown previously that the collagen fibers in heart valves are very mobile since the leaflets can experience considerable shear deformations (Vesely and Boughner, 1989). Such large deformations, particularly in the fibrosa, require a mechanism by which the relatively inextensible fibers are returned into their original starting configuration. The tubular structures of elastin observed in the fibrosa are the likely mechanism for this (Scott and Vesely, 1996). As evident from the contraction of the ventricularis (Vesely and Lozon, 1993), and of isolated elastin, these elastin structures are preloaded in tension. Aortic valve elastin therefore likely acts as a ‘housekeeper’ that restores the collagen fibre geometry back to its original conformation between successive loading cycles, and is therefore critical to proper valve function. Any prosthetic construct intended to simulate the internal micromechanics of the aortic valve should therefore aim to mimic this relationship between elastin and collagen.
Acknowledgments This research was supported initially by a Grant-inAid from the Heart and Stroke Foundation of Ontario, Canada. At that time Dr. Vesely was a Research Scholar of the Foundation. More recently, the work was completed at the Department of Biomedical Engineering at the Cleveland Clinic Foundation under internal funds from the Research Institute. The author would like to acknowledge the assistance of Ms. Marie Malij, supported in part by a Summer Research Student Scholarship from the Natural Sciences and Engineering Research Council of Canada (NSERC), Mr. Gord Niznik, Ms. Joy Dunmore-Buyze, and Dr. Michael Scott. At that time, Dr. Scott was supported in part by an NSERC
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graduate student scholarship. The author is also grateful to Drs. Brian Davis, Evelyn Carew and Derek Boughner for their comments and advice.
References Bashey, R.I., Torii, S., Angrist, A., 1967. Age-related collagen and elastin content of human heart valves. Journal of Gerontology, 20, 203—208. Crissman, R.S., 1987. Comparison of two digestive techniques for preparation of vascular elastic networks for SEM observation. Journal of Electron Microscopy Technique 6, 335—348. Finlay, J.B., Steven, F.S., 1973. The fibrous components of bovine ligamentum nuchae observed in the scanning electron microscope. Journal of Microscopy 99, 57—63. Fung, Y.C., 1981. Biomechanics: Mechanical Properties of Living Tissues. Springer, New York. Gross, L., Kugel, M.A., 1931. Topographic anatomy and histology of the valves in the human heart. American Journal of Pathology, 7, 445—456. Hart, M.L., Beydler, S.A., Carnes, W.H., 1978. The fibrillar structure of aortic elastin. Scanning Electron Microscopy II, 21—28. Kronick, P.L., Sacks, M.S., 1994. Matrix macromolecules that affect the viscoelasticity of calfskin. Journal of Biomechanical Engineering 116 (2), 140—145. Lee J.M., Courtman, D.W., Boughner, D.R., 1984. The glutaraldehydestabilized porcine aortic valve xenograft. I. Tensile viscoelastic properties of the fresh leaflet material. Journal of Biomedical Materials Research, 18(1), 61—77. Lillie, M.A., Chalmers, W.G., Gosline, J.M., 1994. The effects of heating on the mechanical properties of arterial elastin. Connective Tissue Research, 31(1), 23—35. Oxlund, H., Manshot, J., Viidik, A., 1988. The role of elastin in the mechanical properties of skin. Journal of Biomechanics, 21(3), 213—218.
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Roach, M.R., Burton, A.C., 1957. The reason for the shape of the distensibility curves of arteries. Canadian Journal of Biochemical Physiology, 35, 681—690. Scott, M., Vesely, I., 1995. Aortic valve cusp microstructure: elastin’s role. Annals of Thoracic Surgery, 60, S391—S394. Scott, M.J., Vesely I., 1996. Morphology of porcine aortic valve cusp elastin. Journal of Heart Valve Diseases 5(5), 464—471. Song, S.H., Roach, M.R., 1983. Quantitative changes in the size of fenestrations of the elastic laminae of sheep thoracic aorta: studies with SEM. Blood Vessels 20, 145—153. Song, T., Vesely, I., Doughner, D.R., 1990. Effects of dynamic fixation on the shear behaviour of porcine xenograft valves. Biomaterials 11, 191—196. Steven, R.S., Minns, R.J., Thomas, H., 1974. The isolation of chemically pure elastin in a form suitable for mechanical testing. Connective Tissue Research, 2, 85—90. Vesely, I., 1996. Reconstruction of loads in the fibrosa and ventricularis of porcine aortic valves. ASAIO Journal 42, M739—M746. Vesely, I., Boughner, D.R., 1989. Analysis of the bending behaviour of porcine xenograft leaflets and of natural aortic valve material: bending stiffness, neutral axis and shear measurements. Journal of Biomechanics, 22(6/7), 655—671. Vesely, I., Lozon, A., Talman, E., 1993. Is zero pressure fixation truly stress free?, Journal of Thoracic Cardiovascic Surgery 106(2), 288—298. Vesely, I., Lozon A., 1993. Natural preload of aortic valve leaflet components during glutaraldehyde fixation: effects on tissue mechanics. Journal of Biomechanics 26(2), 121—131. Vesely, I., Noseworthy, R., 1992. Micromechanics of the fibrosa and ventricularis of aortic valve leaflets. Journal of Biomechanics, 25(1), 101—113. Viidik, A., Danielson, C.C., Oxlund, H., 1982. On fundamental and phenomenological models, structure and mechanical properties of collagen, elastin and glycosaminoglycan complexes. Biorheology 19, 437—451. Yamazoe, N., Hashimoto, N., Kikuchi, H., Hazama, F., 1990. Elastic skeleton of intracranial cerebral aneurysms in rats. Stroke 21(12), 1722—6.
The role of elastin in aortic valve mechanics Ivan Vesely Department of Biomedical Engineering, The Cleveland Clinic Foundation, Lerner Research Institute, 9500 Euclid Avenue, Cleveland, OH 44195, U.S.A. Received in final form 17 November 1997
Abstract Recent morphologic observations of elastin structures in aortic valves suggest that elastin is mechanically coupled to collagen. Since the mechanical stiffness of elastin is considerably lower than that of collagen, and aortic valves contain relatively little elastin, the mechanical importance of elastin in heart valve function is not clear. We have hypothesized that elastin acts to return the collagen fiber structure back to a resting configuration between loading cycles. The objectives of this research were therefore to elucidate the mechanical relationship between elastin and collagen structures within the aortic valve. To isolate elastin in a morphologically intact state, whole porcine aortic valve leaflets were digested in 0.1 N sodium hydroxide solution (NaOH) at a temperature of 75°C for 45 min. Elastin structures from the fibrosa and ventricularis were tested mechanically, and their loading curves compared to those of the original leaflet layers and to whole cusps. The elastin structures generated very low forces, having an elastic modulus only 0.05% that of the whole tissue. The contribution of elastin to tissue mechanics was significant at low strains and differed between the fibrosa and the ventricularis. Elastin tended to dominate the distensibility curves of the radial ventricularis, but participated very little in the fibrosa. The low but significant tensions produced by the elastin structures of the aortic valve, together with previously observed elastin morphology as well as the measurable preload of elastin, suggest that the purpose of elastin in the aortic valve leaflet is to maintain a specific collagen fiber configuration and return the fibers to this state, once external forces have been released. ( 1998 Elsevier Science Ltd. All rights reserved. Keywords: Elastin; Aortic valve; Collagen; Mechanics; Materials testing; Digestion
1. Introduction The aortic valve cusp is composed of two fibrous layers, the fibrosa and ventricularis, separated by a loose, gelatinous spongiosa (Gross and Kugel, 1931) (Fig. 1). These layers are very mobile and can easily compress and shear during leaflet flexure as the valve opens and closes (Vesely and Boughner, 1989; Song et al., 1990). The fibrosa and ventricularis are also preloaded by virtue of their attachment to each other; the fibrosa under compression and the ventricularis under tension (Vesely and Lozon, 1993; Vesely and Noseworthy, 1992; Vesely et al., 1993). It has been speculated that this preload results from the presence of elastin (Scott and Vesely, 1995). Unlike other highly elastic structures, such as the aorta, the valve cusps contain about 50% collagen and only 13% elastin by dry weight (Bashey et al., 1967). This
would suggest that, relative to collagen, the contribution of elastin to valve leaflet mechanics is minimal. However, during diastolic loading, there is considerable realignment of collagen fibers as the cusps extend beyond 50% strain and recoil elastically. Since collagen on its own is not highly elastic (extensible), we hypothesized that aortic valve elastin is responsible for their elastic recoil. This implies that collagen extends passively through most of its elongation phase. Indeed, recent morphological findings have suggested this mechanism (Scott and Vesely, 1996). The objective of this work was to measure the mechanical properties of aortic valve cusps and their elastin structures to elucidate the specific role of elastin in aortic valve leaflet mechanics, particularly in the recoil mechanism during unloading.
2. Methods * Corresponding author. Tel.: 001 216 445 6671; 216 444 9198; e-mail: [email protected]
fax:
001
0021-9290/98/$19.00 ( 1998 Elsevier Science Ltd. All rights reserved. PII S0021-9290(97)00122-X
The most direct way of measuring the function of elastin in connective tissues is to measure the mechanics
116
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Fig. 1. A cutaway through the leaflet and aortic wall showing the internal configuration of the fibrosa, spongiosa and ventricularis. Near the central portion of the leaflet, the fibrosa exists as a folded sheet that unfolds as the leaflet stretches radially.
of whole specimens, remove all but the elastin, and then retest them. Roach and Burton (1957) used this approach to elucidate the relative contribution of collagen and elastin to the shape of the distensibility curve of arteries. Similarly, Oxlund and Viidik (1988) removed elastin and studied its effects on the biomechanics of skin. Autoclaving is perhaps the best technique for preparing intact elastin with unchanged mechanical properties (Lillie et al., 1994), but unfortunately it cannot be done simultaneously with mechanical testing. Based on amino acid analyses, Finlay and Steven (1973), Steven et al. (1974) and Hart et al. (1978) reported that NaOH and formic acid digestion are very good for purifying elastin. NaOH, in particular, produces elastin with intact inter-molecular cross links making it suitable for mechanical testing. Based on the work of Steven (1974), Song and Roach (1983) and Crissman (1987), we developed a technique to isolate morphologically intact aortic valve elastin. Aortic valve cusps were obtained fresh at the abattoir, typically from cross breeds of Duroc, Hampshire, Yorkshire, and Platrin pigs, usually slaughtered at 5—6 months, at a weight of 235—250 lb. These tissues were digested in 0.1 N NaOH at 75°C. The digestion time was optimized through time/digestion studies during which we examined thin sections stained with Gomori’s Trichrome. We also performed gravimetric studies and hydroxyproline assays, and determined that the optimal digestion time was 45 min. Under SEM, our tissue showed fibers, sheets, and open cells (Scott and Vesely, 1996) (Fig. 2a), structures similar to those reported by Song and Roach (1983), Yamazoe et al. (1990) and Crissman et al. (1987). We further verified the validity of these
Fig. 2. Typical SEM images of elastin structures isolated from aortic valves using NaOH digestion (Scott and Vesely, 1996). Note the wellpreserved fine detail of the elastin structures, particularly (a) a mesh of loose fibers coalescing into a sheet, and (b) an elastin tube exiting the leaflet near its attachment to the aortic wall.
structures by lyophilizing in water and in dimethyl sulfoxide (to prevent ice crystallization) and by critical point drying. In all three techniques, the morphology of elastin was similar. For mechanical testing, 1—3 leaflets from each valve were cut into 5 mm wide circumferential and 10 mm wide radial strips (Fig. 3) and tested in an Instron tensile testing machine (model 1125, Instron Corp., Canton, MA.), while immersed in a bath of Hanks solution heated to 37°C. The tissues were gripped in d320 emery paperlined aluminum grips and preconditioned by repeated stretching at 10 mm/min until reproducible load/elongation curves were attained. The preconditioning load was 5 g for circumferential and 10 g for radial strips. The plotted load/elongation curves were digitized on a Summagraphics tablet (model M11812, Fairfield, Conn.). Since the ‘noise band’ generated by the 100 g load cell was typically 50 mg, manual digitization in some cases smoothed the data. This produced a$0.2 mm uncertainty in gauge length.
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Fig. 3. Image of a typical aortic valve cusp, showing the region from which radial and circumferential test strips re cut. Test strips are always aligned along or transverse to the fibrosal ridges. Because of preconditioning effects, radial strips need to be 10 mm wide.
We took particular care in measuring the gauge length at loads as close to zero as possible. The tissue was clamped between the grips of the Instron and the grips moved together until the tissue was slightly wrinkled. The gain on the load amplifier was set to maximum and the grips slowly moved apart. As the wrinkles in the tissue straightened out, a slight increase in load was observed as the shaft with the grips was withdrawn from the water bath. Since the shaft was cylindrical, its buoyant force decreased linearly with extension until the tissue straightened and began to offer tensile resistance. At that point, the curve began to deviate from the linear plot produced by the buoyant effect. This was the point at which we chose the gauge length. Following testing in the fresh state, the tissue was slackened and the Hanks solution was replaced with preheated 0.1 N NaOH. A thermostatically controlled 1000 W heater maintained the temperature at 75°C during the 45 min of digestion. Following digestion, the digestate was drained, heated saline re-introduced and the digested tissue retested and fractured. Isolated fibrosa and ventricularis were subjected to the same tensile tests and digestions. By carefully grasping the ventricularis and pulling it away from the fibrosa with blunt forceps, the two layers could be separated along the mid-line of the spongiosa. Since it was not possible to measure the thickness of the specimens after digestion, stress could not be determined, and tension (defined as force per unit width of tissue) was used instead. It should also be emphasized that the concept of stress would be meaningless in highly porous structures, like the digested specimens, unless the area fraction of elastin is known. The main purpose of
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this study was to determine the relative mechanics of collagen and elastin, at very low loads. Such low-load experiments could be done under well-controlled conditions only under uniaxial loading. To analyze and compare the loading curves between tissues, these curves needed to be adjusted. We know from previous work (Vesely and Lozon, 1993; Vesely et al., 1993) that once separated from each other, the fibrosa elongates and the ventricularis shrinks, particularly in the radial direction. Once the ventricularis shrinks, its measured extensibility is greater than what it would be when part of the whole leaflet. This is because it was originally preloaded with its operating point slightly to the right of the origin of the extensibility curve. To duplicate this preload, its extensibility curve needs to be shifted to the left. Similarly, since the fibrosa unwrinkled and elongated, its apparent extensibility decreased, and the ‘wrinkled’ phase can be restored by shifting the curve to the right. Such shifting, however, must be done on loading curves where strain is expressed as natural strain, where e"ln [l/l ] (Vesely, 1996). 0 After each incremental shift of the fibrosa and ventricularis, these curves were arithmetically summed together and compared to the curve of the whole tissue. At each step, the ‘goodness-of-fit’ between the shifted and whole tissue curves was evaluated. The minimum of the error value between the whole tissue curve (obtained experimentally) and the layers’ curves (summation of fibrosa and ventricularis) was taken as the optimal shift interval. This shifting process is described in detail in (Vesely, 1996).
3. Results Isolated elastin was found to be extremely extensible, with a very low stiffness (Fig. 4). In the circumferential direction, the stiffness of aortic valve elastin is roughly 1/1700 of that of the whole cusp (2.1 N m~1 for elastin, vs 3.5 kN m~1 near max extension), and its extensibility is well over 100%. Its stiffness is also remarkably linear increasing from 0.31 N m~1 at low loads to 2.1 N/m~1 at high loads. This is consistent with our existing understanding of elastin structures, as reported by Fung (1981). The whole valve, on the other hand, increased its stiffness 1100 times (from 3.1 to 3500 N m~1 at high loads). We also found that elastin structures of the fibrosa and the ventricularis have very different mechanical behavior (Fig. 4). Based on the shape of the tension/strain curves and the low fracture strength, very little elastin exists within the fibrosa. The fracture tension of the fibrosal elastin was 0.35 and 0.14 N m~1 in the circumferential and radial directions, respectively, whereas elastin from the ventricularis had a mean fracture tension almost an order of magnitude greater, at 2.94 and 2.81 N m~1 (Table 1).
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Fig. 4. Original data curves for all the specimens tested in this study, converted to tension vs strain. These curves show the relative variability of the data, as well as the gross differences between the groups. The fresh tissues were stretched to arbitrary loads, and digested tissues were stretched to failure.
While elastin structures generate forces many times lower than collagen, they are not negligible. The relative contribution of elastin can be estimated through a comparison of their tension/strain curves, once these curves were appropriately shifted. The optimal shift values were e"0.25 and e"0.11 for the radial fibrosa and the ventricularis, respectively (Fig. 5). This means that the
fibrosa needed to be shifted by 0.25 to the right and the ventricularis by 0.11 to the left. Similarly, for circumferential tension curves, the optimal shift was at 0.11 for the fibrosa and 0.014 for the ventricularis (Fig. 5). Examining the shifted tension/strain curves, it can be seen that in the radial direction (Fig. 5d), the ventricularis appears to dominate the elastic response in the low-strain
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Fig. 5. Diagrammatic representation of the curve shifting process: (a) The original functions for the whole cusp, the fibrosa and the ventricularis, tested in the circumferential direction. Note that the fibrosa should be shifted to the right and the ventricularis to the left, before summing the curves together and comparing with the curve from the whole tissue. (b) Plots showing the shape of the circumferential fibrosa and ventricularis curves after optimal shifting. The fibrosa and the ventricularis were shifted by 0.25 and 0.11 strain units to the right and left, respectively. (c) The original functions for the whole cusp, the fibrosa and the ventricularis, tested in the radial direction. Note that the fibrosa should be shifted to the right and the ventricularis to the left before summing them together and comparing with the whole tissue. (d) Plots showing the shape of the radial fibrosa and ventricularis curves after optimal shifting. The fibrosa and the ventricularis were shifted by 0.11 and 0.014 strain units to the right and left, respectively.
region. At these low strains, the fibrosa does not contribute any tension since it is highly folded and is simply unfurling its coils. In the circumferential direction (Fig. 5b), a similar process occurs, but to a lesser extent. Below strains of 0.15, the fibrosa has little effect, between 0.15 and 0.25, the fibrosa and the ventricularis contribute almost equally, and above strains of 0.25 and loads of 2.0 N m~1, the fibrosa begins to dominate. Unlike the loading curves of the fibrosa and ventricularis that had to be reconstructed mathematically, the loading curves of elastin structures were adjusted
from direct measurements. Because the same tissue strip was tested before and after digestion without removal from the tissue grips its gauge length, and hence shrinkage, was measured directly. The isolated elastin structures shrank by 10—30% during digestion (Table 2), indicating that they were preloaded in tension when connected to collagen. Once the elastin curves were shifted by the appropriate amounts, it can be seen that in the fibrosa, the majority of the tension is generated by collagen (Fig. 6). This is because the elastin can generate only about 0.1 N m~1 of tension at physiological strains. In the ventricularis, however, the elastin structures
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dominate the mechanical response in the radial direction, while in the circumferential direction, the loads are shared almost equally between elastin and collagen up to 0.25 strain. The elastin plots of whole cusps are inconclusive, as different amounts of contraction likely took place in the elastin of the fibrosa and the ventricularis.
Table 1 Fracture parameters for elastin isolated from whole cusps and layers
Circumferential
Whole
Fibrosa
Ventricularis
Radial
Whole
Fibrosa
Ventricularis
N Mean S.E.M. N Mean S.E.M. N Mean S.E.M. N Mean S.E.M. N Mean S.E.M. N Mean S.E.M.
Fracture tension (N m~1)
Fracture strain(%)
15 2.72 0.29 7 0.35 0.06 8 2.94 0.29 8 1.65 0.36 10 0.14 0.01 10 2.81 0.30
15 143 10 7 101 12 8 152 12 8 127 17 10 147 13 10 170 12
4. Discussion In this study, we have characterized porcine aortic valve elastin and estimated its relative contribution to the mechanics of the fibrosa and ventricularis. Both the mechanical properties of elastin, as well as the changes in gauge length following digestion, confirm our hypothesis that elastin structures impose tensile forces on collagen fibers during valve unloading. In order to accept the results of this work, however, certain assumptions have to be made. First, we must assume that the mechanical behavior of tissues measured in vitro is representative of their behavior in vivo, under biaxial loading conditions. Experience has shown that while absolute values of elastic moduli and extensibility may differ from real, in vivo conditions, standardized uniaxial tensile tests, such as these, are extremely sensitive to differences in mechanics between test groups. Certainly, the poor aspect ratio of the radial tissue strips can be one source of error. The radial strips were chosen wider than the circumferential because strips of tissue narrower than 7 mm have been shown to fail during preconditioning (Lee et al., 1984). Although the low aspect ratio does not permit uniform uniaxial loading, these effects were present in both fresh and digested specimens, and would not likely affect the conclusions drawn regarding load sharing between elastin and collagen. The shrinkage of the digested tissues suggests denaturation. Mature elastin, however, is highly stable at elevated temperatures (Lillie et al., 1994) and is unlikely to be damaged, particularly since fine structures were
Table 2 Mean gauge length of fresh and digested aortic valve cusps and their components Difference
Circumferential
Whole
Fibrosa
Ventricularis
Whole
Radial
Fibrosa
Ventricularis
N Mean S.E.M. N Mean S.E.M. N Mean S.E.M. N Mean S.E.M. N Mean S.E.M. N Mean S.E.M.
Fresh (mm)
Digested (mm)
15 8.2 0.42 7 8.9 0.59 8 7.4 0.81 8 5.3 0.28 10 5.6 0.42 10 4.0 0.43
15 8.0 0.40 7 7.9 0.63 8 6.4 0.75 8 4.7 0.53 10 4.0 0.33 10 3.4 0.41
% strain
Natural strain
Significance
0.024
NS
11
0.10
p(0.088
14
0.13
p(0.0035
11
0.10
NS
29
0.25
p(0.00001
15
0.14
p(0.00001
2.4
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well preserved (Fig. 2). The longitudinal shrinkage therefore likely resulted from the release of some residual stress upon the removal of collagen, and is consistent with the observations of Oxlund et al. (1988).
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Perhaps the most important biomechanical effect of isolating elastin is that one removes the collagen fiber bundles that fill spaces within the elastin structures. Consequently, during stretching, the porous elastin structure
Fig. 6. Plots of the functions describing the mean behavior of fresh and digested tissues. Note the widely varying contribution of elastin to the function of the valve layers. However, while these plots can be used to determine the relative contribution of elastin to the behavior of the fibrosa and the ventricularis, it cannot be done for the whole tissue. The elastin curves for the whole tissue represent elastin from both the fibrosa and the ventricularis, which exist in some mixed form of preload. Since these elastin curves represent preloaded materials, they have not been shifted and therefore cannot be used for superposition.
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can collapse much more than it could have when the voids were filled with collagen. This likely increases the extensibility and compliance of the elastin. It is interesting to note, however, that the elastin of the radial ventricularis (Fig. 6) follows the original ventricularis curve very closely to almost 50% strain, suggesting that the elastin sheets of the ventricularis are flat, and do not have many voids or undulations that could collapse. This is consistent with our morphological observations (Scott and Vesely, 1996). Finally, the mechanical effects of the proteoglycans in the ground substance of the valve were not considered in this analysis. Proteoglycans bind intimately with collagen fibers and could significantly reinforce the collagen fiber matrix. The loads carried by ‘collagen’ should therefore be considered as a summation of loads distributed between collagen and proteoglycans. However, proteoglycans typically contribute mechanically only during dynamic loading and have minimal contribution statically. Indeed, Viidik et al. (1982), and more recently Kronick and Sacks (1994) have shown no significant effects of proteoglycan extraction on the static mechanical properties of biological tissues. In the context of the above assumptions, the results of this study offer an improved understanding of the internal micromechanics of aortic valve cusps and the functional role played by elastin structures. First of all, the mechanical data confirm the low proportion of elastin in heart valve leaflets (Bashey et al., 1967). Because of their porosity, these elastin structures have a maximal stiffness of only 10 kPa, more than an order of magnitude lower than the 400 kPa of the relatively pure elastin from ligamentum nuchae (Fung, 1981) (stiffness of valve elastin was estimated from Fig. 4, assuming a thickness of 0.5 mm). As the ventricularis is stretched in the radial direction, the dominant restorative force is the tension produced by elastin. This is not surprising, since the ventricularis must undergo extensions of up to 60% strain and collagen is relatively inextensible. The collagen is likely organized in a highly wavy configuration and uncoils passively through most of the extension of the ventricularis. Once extended fully, the collagen takes over the load and limits further extension. This was suggested by us previously (Vesely and Noseworthy, 1992) and has now been verified through direct mechanical testing. In the circumferential direction, however, collagen appears to play a more significant role, sharing load roughly equally with elastin up to the transition phase (&20% strain). In the fibrosa, elastin plays a minor role, both radially and circumferentially. In the radial direction, the fibrosa has a macroscopic waviness so that it can stretch to large strains passively until ‘lock-up’. Because of the extremely low mechanical contribution of fibrosal elastin (Fig. 6), the fibrosa likely relies on the ventricularis to pull it back into its folded shape.
The function of elastin is therefore highly dependant on its configuration and relative content in the given material. Unlike the low-modulus ‘elastin’ phase of the aorta (Roach and Burton, 1957), aortic valve elastin can have (i) a minimal contribution to mechanics, as in the fibrosa; (ii) can participate equally with collagen, as during initial stretching of the ventricularis circumferentially; and (iii) can also totally dominate the mechanics at the early phase, as in the radial ventricularis. It is important to note that elastin content alone does not dictate mechanics, but rather its organization relative to collagen. The ventricularis contains a considerable amount of sheet elastin (Scott and Vesely, 1996) with loading curves that appear identical radially and circumferentially (Fig. 4 and 6). Since the mechanics of the whole ventricularis are not identical in the radial and circumferential directions, it must be the amount of collagen or the types of connections between the elastin and collagen structures, that are responsible for this anisotropy, not the mechanics of the elastin itself. We have shown previously that the collagen fibers in heart valves are very mobile since the leaflets can experience considerable shear deformations (Vesely and Boughner, 1989). Such large deformations, particularly in the fibrosa, require a mechanism by which the relatively inextensible fibers are returned into their original starting configuration. The tubular structures of elastin observed in the fibrosa are the likely mechanism for this (Scott and Vesely, 1996). As evident from the contraction of the ventricularis (Vesely and Lozon, 1993), and of isolated elastin, these elastin structures are preloaded in tension. Aortic valve elastin therefore likely acts as a ‘housekeeper’ that restores the collagen fibre geometry back to its original conformation between successive loading cycles, and is therefore critical to proper valve function. Any prosthetic construct intended to simulate the internal micromechanics of the aortic valve should therefore aim to mimic this relationship between elastin and collagen.
Acknowledgments This research was supported initially by a Grant-inAid from the Heart and Stroke Foundation of Ontario, Canada. At that time Dr. Vesely was a Research Scholar of the Foundation. More recently, the work was completed at the Department of Biomedical Engineering at the Cleveland Clinic Foundation under internal funds from the Research Institute. The author would like to acknowledge the assistance of Ms. Marie Malij, supported in part by a Summer Research Student Scholarship from the Natural Sciences and Engineering Research Council of Canada (NSERC), Mr. Gord Niznik, Ms. Joy Dunmore-Buyze, and Dr. Michael Scott. At that time, Dr. Scott was supported in part by an NSERC
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graduate student scholarship. The author is also grateful to Drs. Brian Davis, Evelyn Carew and Derek Boughner for their comments and advice.
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