Br. J. exp. Path. (1977) 58, 572
THE EFFECT OF CALCIUM ON THE MECHANICAL BEHAVIOUR OF AORTA MEDIA ELASTIN AND COLLAGEN R. J. MINNS AND F. S. STEVEN
From the Department of Engineering Science, University of Durham, South Road, Durham. and the Department of Medical Biochemistry. University of Manchester Received for publication June 16, 1977
Summary.-Mechanical tests were conducted on rings of pig thoracic aorta that had been treated with calcium ions to evaluate their effect on aorta media coliagen and elastin. There was an increase in stiffness just before rupture and a decrease in storage energy when calcium was introduced into intact rings, but there was no significant difference in mechanical properties when it was introduced into the elastin framework of the media. The calcium was able to bind to the intact aorta media far more readily than to the elastin alone, suggesting that the collagen and muscle framework were far more susceptible to the introduction of calcium than elastin. The calcium content rose along the length of the aorta, as did the collagen content, whilst the elastin content fell, which was also reflected in the change in mechanical properties along the aorta. THE relation of the structure to the function of the arterial wall has fascinated scientists for many years. Burton (1954) reviewed publications on the subject up to the early 1950s and pointed out the importance of determining the mechanical behaviour of the elastin, collagen and smooth muscle before the physiological functioning of their interaction could be understood. He, with Learoyd and Taylor (1966), have attributed the viscositv of the aorta to the smooth muscle present and believed the main structural components of the wall to be the elastin and collagen fibrous networks. The role of these two fibrous networks has been evaluated in other tissues (Minns, Soden and Jackson. 1973) and it is generally accepted that the modulus at low extensions in the aorta is reflected by the elastin present, whilst the collagen probably prevents the wall from distending beyond the yield point of elastin (Harkness, Harkness and MIcDonald, 1957; Sharma, 1974). However, it would appear
that the collagen and elastin are involved in a complex structural relationship, the strength of the collagen network depending on the integrity of the elastin (Coulson, Weissman and Carnes, 1965). Most of the elastin fibres in the vein are continuous longitudinally but discontinuous circumferentially, whilst the collagen fibres form another network structure in a crimped state (Azuma and Hasegawa, 1973). In the aorta, tangential stressing is resisted by this crimped collagen network, while the elastin distributes the distending forces uniformly throughout the wall (Wolinsky and Glagov, 1964). Certainly, in the pig aorta, circumferentially aligned strips are mechanically stronger than longitudinal strips (Wegner and Feder, 1976) which probably reflects the circumferentiallv preferred collagen network within the aorta wall. The individual mechanical roles of the elastin and collagen frameworks within the aorta media have been difficult to investigate, since treatment to remove one
Address for correspondence: Dr R. J. Minns, Department of Engineering Science, University of Durham, South Road, Durham, DH1 3LE England.
573
MECHANICAL BEHAVIOUR OF THE AORTA
of the fibrous components produces changes in the other. Elastase has been used to evaluate the mechanical behaviour of the "collagen residue" (Coulson et al., 1965), and proteolytic enzvmes have been used to estimate the contribution of aortic elastin to the mechanical behaviour of the whole aorta (Coulson, 1971). Using a technique we developed (Steven, Minns and Thomas, 1974) to produce chemically pure elastin from bovine ligamentum nuchae, mechanical tests were conducted on pig aorta rings to evaluate the mechanical behaviour of aortic elastin. Changes in the gross structural components of the arterial wall are known to alter the mechanical properties of the aorta (Minns and Steven, 1976) in particular in the region of lesions induced by copper deficiency (Coulson and Carnes, 1962) and by trauma (Kivity andCollins, 1974). Calciumplaques developed in the advanced stages of arteriosclerosis show microscopic structural changes in the fibrous components of the aorta wall (Kadar et al., 1975). Therefore a technique was developed to introduce calcium into the media of normal pig aorta and aortic elastin, and to observe its effect microscopically and mechanicaRy. MATERIALS AND METHODS Source of Aortae.-The aortic rings
were cut
from the region distal to the thoracic arch of freshy slaughtered pigs and were cleaned of adheringtissue.Thelength(L) in cm from the thoracic arch and the internal diameter (D) in mm of each ring were recorded and plotted against each other. The graph up to 25 cm length was linear and the relationship between D and L could be expressed from the data of 5 pigs, as
L = 46-7 - 2-63D A gravimetric method was employed for determinig the cross-sectional area of each ring. With a specific gravity for aorta of 1-06, the area (A) was related to the weight (W) in g and diameter (D) in mm by the relationship A = 28-9 (W/D) mM2 Biochemica treatment.-To observe the behaviour of the elastin in the aorta, the ground substance and collagen were removed by alternate treatments 'With guanidium chloride (G) and purified collagenase (C), described in detail by Steven et al. (1974) as GCGCG pretreatment. Calciujm was bound to the elastic lamellae by introducing rings of purified aortic elastin into 5 ml of 4m CaC12 for 3 h twice, the rings being washed in distilled water between and after treatments. Calcium was also introduced into untreated aorta rings by soaking for 1 h in 02 X CaCl2 twice and washed with distilled water. The weaker treatment was used since possible damage to the collagen in strengths of CaCl2 greater than 2m has been reported by Gustavson (1956). Sixteen rings taken at random from the a animals were treated by one of the techniques described and 32 were selected at random as control rings, a total of 80 rings.
Cut, weigh and number ring Test mechanically, treated ring to rupture
I Air dry wt
Air dry wt
Acetone dry wt
Acetone dry wt
Hydrolysis, 1 ml of 6N-
HCI, 72 h at
Ca-- content using
analytical standards for Calcium with an automatic atomic absorption spectrophotometer.
105c
Treat with 0-1
X
N'aOH
I hour at 100'
Remove HCI
wash
Hydroxyproline analysis
Acetone dry
to determine collagen content by method of Woessner (1961)
'Weigh residue
Determine elastin content by method of Lansing et al. (1952) Fig. 1 -Procedure used to determine the elastin, collagen and calcilnm contents of the aorta rings tested.
5744
R. J. MINNS AND F. S. STEVEN
Mlechanical pruperties of aortic rings.-AII the rings were tested im 0-9 % NaCl at 200 in an Instron type TM 1112 table model tensile testing machine (Minns et al., 1973). The specimen grips were slightly modified to contain L-shaped hooks, the rings being slipped over these before extension. Zero extension was recorded when the ring sides became parallel between the hooks, the distance between the hook rod centres being zero extension. The rings were elongated at a constant rate of 2 mm/min, extension being recorded by crosshead displacement and load simultaneously recorded by a sensitive load cell fixed to the upper specimen hook. The stress was calculated by dividing the load obtained (in Newtons) by the initial cross-sectional area (in m2), the strain being the recorded extension divided by the distance between the hooks at zero extension. Scanning electron microscopy.-After the mechanical tests were conducted, specimens of interest were observed in the scanning electronmicroscope. A very careful dehydration procedure was used, the specimens being immersed in increasing strengths of acetone (50, 60, 70, 80, 90, 95, 100%, 20 min at each step) followed by critical-point drying using C02 as the drying liquid in a Polaron E 3000 critical-point drying apparatus. The dried specimens were cut and mounted in different orientations to show rupture and
structural details on marked i-inch aluminium stubs and coated with gold/palladium in a Polaron E 5000 sputter coating unit to produce a coating 150-200 A thick. The coated specimens were examined in a Cambridge Stereoscan S4-10 scanning electronmicroscope at an accelerating voltage of 20kV and filament current of 2-7 amps, the results permanently recorded with an Exacta %X5000 camera on Ilford FP4 135 roll film. Determination of calcium, coUagen and eiastin content.-The techniques used to determine the calcium, collagen and elastin contents of the rings after the mechanical tests were conducted are shown in Fig. 1. The calcium contents were expressed as ja/g dr-y wt of tissue, whilst the collagen and elastin contents were defined as % dry wt of the tissue samples analysed. RESULTS
Stress-strain curves of the aorta strips tested mechanicallv are shown in Fig. 2. Two factors appear to emerge from the observations of the effect of calcium ions on the mechanical properties of intact aorta media. First, there is an increase in the final modulus with only a slight decrease in the breaking stress and
3
Ca2+
_2_ x
E z 0 U)
0
40
60
Strain (%) Fig. 2.-Stress-strain behaviour of the rings from the thoracic
aortae of
pigs.
MECHA-NICAL BEHAVIOUR OF THE AORTA
strains and, secondlv, a decrease in the storage energy or area under the stressstrain curve. The calcium introduced into the elastin framework had the effect of stiffening the matrix of the wall but could not extend the network more than the GCGCG-treated rings. The storage energy, final moduli, breaking stress and
575
breaking strains of the 4 test svstems are summarized in the Table. The ultimate breaking stress along the length of the aorta increased for the intact aorta, and for rings treated with calcium it remained fairly constant (Fig. 7). The calcium content within the rings increased down the aorta up to 18 cm
Fig. 3.-Scanning electronmicrograph of the ruptured end of a control aortic ring; the internal surface is on the bottom right. x 60. Fig. 4.-Scanning electronmicrograph of the lamellae in the region of the rupture of a control aortic ring, showing some of the lamellae separating, exposing the inter-lamella structures. x 120. Fig. 5.-Scanning electronmicrograph of the ruptured end of an aortic ring treated with calcium. x 60.
Fig. 6.-Scanning electronmicrograph of the lamellae within an aortic ring treated with calciujm. x 120.
5w7 6
R. J. MINNS AND F. S. STEVEN
0 x
E z S S S
,
ca2+
S b. .0 C
ES2
a
0
5 10 15 L - ditauorg ta (ams) Fig. 7. Variation of the ultimate breaking stress (or maximum recorded load divided by the initial cross-section area) along the length of the pigs thoracic aorta. L = 0 being the top of the aortic arch.
from the thoracic arch, with an approximate twentv-fold increase in the values of the calcium-treated rings over the control but onlv double when calcium was introduced into the elastin-prepared rings. The values of the GCGCG-treated control rings before calcium treatment had apparent higher calcium content than the control values of the intact rings because the values were estimated for the tissue dry wt after treatment for the removal of the non-elastin components (Fig. 8). The dry wt elastin content fell, whilst the collagen content rose slightly along the length of the aorta, the total fibrous content (elastin and collagen) falling from 72 to 57%0 dry wt (Fig. 9). W7hen the ruptured rings were observed in the scanning electronmicroscope, the intact rings broke "cleanly" leaving an even fracture surface (Fig. 3) with little disruption of the lamellae (Fig. 4). Rings treated with calcium and ruptured exhibited a contrasting fracture surface, the surface breaking unevenly, leaving a "ragged" torn surface (Fig. 5), the individual lamellae appearing to be grossly disrupted (Fig. 6). The morphological findings from scanning electronmicroscopy are summarized in Fig. 10 which shows diagramatically the plan and internal views of the rupture phenomena.
200
150-
0
E 1 100
_
0
E 13 a
A
U
5oF AA ( 0
DOw
10 L - length aong Aorta (ams)
20
Fig. 8.-Variation of calcium content along the aorta. 0-Control, A-GCGCG-treated, A-GCGCG and Ca++, *-Ca++-treated intact rings.
577
MECHANICAL BEHAVIOU'R OF THE AORTA
40 .C
a.0
6 12 1 L - ngth along Aorta (cms) Fig.9. Varation of elastin and collagen content along the aorta. C-Elastin, 0-Collagen. O
TABLE.--Summary of Storage Energy, Final Modulus, Breaking Stress, and Breaking Strain of the Test Systems Breaking stress Treatment Control Ca++ GCGCG Ca-GCGCG -
(N/M2)
Breaking strain
(°h)
x 106
2 -916 : 0 -25 2-537 T 0-21 0-368 0-015
0-138 0: 0-012
62-0 2 2-1
60-3 2 2-0
43.4 = 1-2
46-4 = 1-0
DISCUSSION
It is clear from the analysis of the calcium content in the intact and elastinprepared media that the calcium introduced binds to the collagen framework and not to the elastic tissue. Collagen contains many carboxyl groups, and the positive calcium ions are bound by ionic interaction of aspartic and glutamic acid residues within the polypeptide chains of the polymeric collagen fibril to these sites. Elastin has very few carboxyl groups; thus there is little or no binding of calcium ions by ionic interaction, and even though considerable amounts of calcium ions were introduced into the
Stored energy (N/M2) X 104 48-9 - 4-1
31-25 2-7 3.3 2- 0-4 1-2 = 1-0
Final modulus (N/M2) X 106 15-0
28-0 3-0 1 -2
elastin framework there was little effect on the mechanical properties and only approximatelv double the amount of detectable calcium. In the intact media which contains collagen and elastin, the binding of calcium greatly increased by equilibrium with a low concentration of calcium chloride. The ionic binding of calcium must therefore be associated with the carboxyl and sulphate groups of the collagen fibrils and proteoglycans respectively. Although the ground substance plays a small role in the mechanical behaviour of the aorta and is particularly related to its time-dependent response (Minns et al., 1973), the collagen fibres are
578
R. J. MIN"NS ANYD F. S. STEVEN
lagen content; however, when calcium is introduced, the breaking stress becomes lower than the intact aorta distallv. Clinically, lesions associated with calcium plaques and intimal thickening (Kadar et al., 1975) give rise to ruptures in the distal portion of the thoracic aorta, possibly related to the higher collagen content distally and hence the ability to bind more calcium, making the aorta wall more brittle and susceptible to rupture. Introduction of calcium has a dramatic effect on the morphology at the rupture site. In the normal intact aorta rings, the rupture was a clean break through the wall; the lamellae were all ruptured at the same level. The clean breaks are similar to those observed clinicallv in traumatic rupture of normal aortae (Sevitt, 1977); presumably the tensile strength of the collagen/elastin framework is exceeded at the same level through the wall. In the case of the calcium-treated aorta, the lamellae were very disrupted and the appearance of the rupture suggested b breakage at different levels and sites Fig. 10.-Sketches of the mode of failure of through the wall. The collagen/elastin the specimens. (a) The intact control rings broke cleanly through the wall from the framework appears to be structurallv inside, whilst (b) the calcium-treated specidisrupted similar to that observed within mens, although failing from the inside, showed gross disruption of the lamellae arteriosclerotic lesions (Kadar et al., 1975), during failure. the collagen fibrils being mechanicallv more brittle at sites where calcium is significantly related to the load and bound, producing the uneven rupture extension properties of the wall. appearance observed. The introduction of calcium within the We wish to thank Professor G. R. media had the effect of increasing the final modulus or stiffness (slope of the Higinson for the mechanical testing stress-strain curve) near the point of facilities, Dr G. Williams and ir D. S. rupture in both the elastin preparation and Page, Department of Pathology, Unithe intact specimen. In the calcium- versitv of Manchester for the scanning treated intact specimens the breaking electronmicroscope facilities and 3irs J. stress decreased. These two characteris- Watkins in the Department of Medical tics of increased stiffness and lowered Biochemistrv, Universitv of Manchester. ultimate tensile stress together with a We would also like to thank Mliss K. fall in storage energy suggest the media McConnon for conducting the calcium becomes "brittle", whilst in the elastin- analysis, and Dr E. G. Cleary for helpful prepared specimens the ultimate tensile discussions. stress doubled when calcium was introREFERENCES duced. The increase in breaking stress as M. (1973) Distensibility T. & HASEGAWA, -AzumA, one progresses distally along the aorta of the Vein: from the Architectural Point of may be reflected in the increase in colView. Biorheology, 10, 469.
MECHANICAL BEHAVIOUIR OF THE AORTA BURTON-, A. C. (1954) Relation of Structur to Function of the Tisues of the WaIl of Blood Vessels. Physil. Ret., 34, 619. Couu_oN, W. F. & CARNEs, W. H. (1962) Cardiovascular Studies on Copper-deficient Swine. II. Mechanial Properties of the Aorta. Lab. Inres., 11, 1316. CouisoN, W. F., WIssmAN, N. & CANs, W. H. (1965) Cardiovascular Studies on Copper-deficient Swine. VIIIM echanil Properties of Aortic and Dermal Collagen. Lab. Inved., 14, 303. CouisoN, W. F. (1971) The Effect of Proteolytic Enzymes on the Tensile Strength of Whole Aorta and Isolated Aortie Elastin. Biochem. bi;phys. Ada., 237, 378. GusTAvsoN, K. M. (1956) In The Chemistry and Reacirity of ColUagen. New York, London: Academic Press, p. 179. HIARx ss, M. L. R., HAERNEsS, R. D. & MCDONALD DA. (1957) The Collagen and Elastin Content of the Arterial Wall in the Dog. Proc. roy. Soc. (Lond.), Series B, 146, 541. KA A , A., BIrARI-VARGA, IL, Gmmo, S. & JILLINKK, H. (1975) Composition and Maeromolecular Structure of Intima in Normal and Arteriosclerotic human aorta. Arterial Wall, 3, 3. KIvITY, Y. & COTmL s, R. (1974) Nonlinear Wave Propagation in Viscoelastic Tubes: Application to Aortic Rupture. J. Biomeek., 7, 67. LANSNG, A. I., RosFNTmL, T. B., ALEX, Mx & DExPSEY, E. W. (1952) The Structure and Chemical Characteristics of Elastic Fibres as
579
Reveaed by Elastase and Electron Microscopy. Anat. Roe., 114, 555. LzwoYD, B. M. & TAYLOR, M. G. (1966) Alterations with Age m the Vco-elastic Properties of Human Arial Walls Circulation Res., 18, 278. MSis, R. J., SODEN, P. D. & JAcxsoN, D. S. (1973) The Role of the Fibrous Components and Ground Substance in the Mecanial Properties of Biologial Tises. A Preliminay Investigation. J. BiomenA., 6, 153. MINs, R. J. & STmvzx, F. S. (1976) The Tensile Properties of Developing Fetal Elastic tissue. J. Biomnc., 9, 9. Sxvrrr, S. (1977) The lilehani of Traumatic Ruptu,re of the Thoracic Aorta. Br. J. Surg., 64, 166. SgARMA, M. G. (1974) Viscoelastic Behaviour of Conduit Arteries. Bioreogogy, 11, 279. STEVxN, F. S., MiNs, R. J. & THotAs, H. (1974) The Isolation of Chemically Pure Elastics in a Form Suitable for ehanial Testing. Conn. Ti. Res., 2, 85. WGNaaR, W. & Fjm)R, H. (1976) Zur Gewebsmechanik der Aortenwand des Schweines (Some Mechanical Propertes of the Pig Aorta) Res. Ezp. Med., 168, 129. WOXSSNEP, J. F. (1961) The Determination of Hydroxyproline in Tissue and Protein Samples Containing Small Proportions of this Imino Acid. Arch. Biochem. Biophys., 93, 440. WoLNsKY, H. & GYAGOV, S. L. (1964) Structural Basis for the Static Mechanical Properties of the Aortic Media. Circulation Res., 14, 400.
THE EFFECT OF CALCIUM ON THE MECHANICAL BEHAVIOUR OF AORTA MEDIA ELASTIN AND COLLAGEN R. J. MINNS AND F. S. STEVEN
From the Department of Engineering Science, University of Durham, South Road, Durham. and the Department of Medical Biochemistry. University of Manchester Received for publication June 16, 1977
Summary.-Mechanical tests were conducted on rings of pig thoracic aorta that had been treated with calcium ions to evaluate their effect on aorta media coliagen and elastin. There was an increase in stiffness just before rupture and a decrease in storage energy when calcium was introduced into intact rings, but there was no significant difference in mechanical properties when it was introduced into the elastin framework of the media. The calcium was able to bind to the intact aorta media far more readily than to the elastin alone, suggesting that the collagen and muscle framework were far more susceptible to the introduction of calcium than elastin. The calcium content rose along the length of the aorta, as did the collagen content, whilst the elastin content fell, which was also reflected in the change in mechanical properties along the aorta. THE relation of the structure to the function of the arterial wall has fascinated scientists for many years. Burton (1954) reviewed publications on the subject up to the early 1950s and pointed out the importance of determining the mechanical behaviour of the elastin, collagen and smooth muscle before the physiological functioning of their interaction could be understood. He, with Learoyd and Taylor (1966), have attributed the viscositv of the aorta to the smooth muscle present and believed the main structural components of the wall to be the elastin and collagen fibrous networks. The role of these two fibrous networks has been evaluated in other tissues (Minns, Soden and Jackson. 1973) and it is generally accepted that the modulus at low extensions in the aorta is reflected by the elastin present, whilst the collagen probably prevents the wall from distending beyond the yield point of elastin (Harkness, Harkness and MIcDonald, 1957; Sharma, 1974). However, it would appear
that the collagen and elastin are involved in a complex structural relationship, the strength of the collagen network depending on the integrity of the elastin (Coulson, Weissman and Carnes, 1965). Most of the elastin fibres in the vein are continuous longitudinally but discontinuous circumferentially, whilst the collagen fibres form another network structure in a crimped state (Azuma and Hasegawa, 1973). In the aorta, tangential stressing is resisted by this crimped collagen network, while the elastin distributes the distending forces uniformly throughout the wall (Wolinsky and Glagov, 1964). Certainly, in the pig aorta, circumferentially aligned strips are mechanically stronger than longitudinal strips (Wegner and Feder, 1976) which probably reflects the circumferentiallv preferred collagen network within the aorta wall. The individual mechanical roles of the elastin and collagen frameworks within the aorta media have been difficult to investigate, since treatment to remove one
Address for correspondence: Dr R. J. Minns, Department of Engineering Science, University of Durham, South Road, Durham, DH1 3LE England.
573
MECHANICAL BEHAVIOUR OF THE AORTA
of the fibrous components produces changes in the other. Elastase has been used to evaluate the mechanical behaviour of the "collagen residue" (Coulson et al., 1965), and proteolytic enzvmes have been used to estimate the contribution of aortic elastin to the mechanical behaviour of the whole aorta (Coulson, 1971). Using a technique we developed (Steven, Minns and Thomas, 1974) to produce chemically pure elastin from bovine ligamentum nuchae, mechanical tests were conducted on pig aorta rings to evaluate the mechanical behaviour of aortic elastin. Changes in the gross structural components of the arterial wall are known to alter the mechanical properties of the aorta (Minns and Steven, 1976) in particular in the region of lesions induced by copper deficiency (Coulson and Carnes, 1962) and by trauma (Kivity andCollins, 1974). Calciumplaques developed in the advanced stages of arteriosclerosis show microscopic structural changes in the fibrous components of the aorta wall (Kadar et al., 1975). Therefore a technique was developed to introduce calcium into the media of normal pig aorta and aortic elastin, and to observe its effect microscopically and mechanicaRy. MATERIALS AND METHODS Source of Aortae.-The aortic rings
were cut
from the region distal to the thoracic arch of freshy slaughtered pigs and were cleaned of adheringtissue.Thelength(L) in cm from the thoracic arch and the internal diameter (D) in mm of each ring were recorded and plotted against each other. The graph up to 25 cm length was linear and the relationship between D and L could be expressed from the data of 5 pigs, as
L = 46-7 - 2-63D A gravimetric method was employed for determinig the cross-sectional area of each ring. With a specific gravity for aorta of 1-06, the area (A) was related to the weight (W) in g and diameter (D) in mm by the relationship A = 28-9 (W/D) mM2 Biochemica treatment.-To observe the behaviour of the elastin in the aorta, the ground substance and collagen were removed by alternate treatments 'With guanidium chloride (G) and purified collagenase (C), described in detail by Steven et al. (1974) as GCGCG pretreatment. Calciujm was bound to the elastic lamellae by introducing rings of purified aortic elastin into 5 ml of 4m CaC12 for 3 h twice, the rings being washed in distilled water between and after treatments. Calcium was also introduced into untreated aorta rings by soaking for 1 h in 02 X CaCl2 twice and washed with distilled water. The weaker treatment was used since possible damage to the collagen in strengths of CaCl2 greater than 2m has been reported by Gustavson (1956). Sixteen rings taken at random from the a animals were treated by one of the techniques described and 32 were selected at random as control rings, a total of 80 rings.
Cut, weigh and number ring Test mechanically, treated ring to rupture
I Air dry wt
Air dry wt
Acetone dry wt
Acetone dry wt
Hydrolysis, 1 ml of 6N-
HCI, 72 h at
Ca-- content using
analytical standards for Calcium with an automatic atomic absorption spectrophotometer.
105c
Treat with 0-1
X
N'aOH
I hour at 100'
Remove HCI
wash
Hydroxyproline analysis
Acetone dry
to determine collagen content by method of Woessner (1961)
'Weigh residue
Determine elastin content by method of Lansing et al. (1952) Fig. 1 -Procedure used to determine the elastin, collagen and calcilnm contents of the aorta rings tested.
5744
R. J. MINNS AND F. S. STEVEN
Mlechanical pruperties of aortic rings.-AII the rings were tested im 0-9 % NaCl at 200 in an Instron type TM 1112 table model tensile testing machine (Minns et al., 1973). The specimen grips were slightly modified to contain L-shaped hooks, the rings being slipped over these before extension. Zero extension was recorded when the ring sides became parallel between the hooks, the distance between the hook rod centres being zero extension. The rings were elongated at a constant rate of 2 mm/min, extension being recorded by crosshead displacement and load simultaneously recorded by a sensitive load cell fixed to the upper specimen hook. The stress was calculated by dividing the load obtained (in Newtons) by the initial cross-sectional area (in m2), the strain being the recorded extension divided by the distance between the hooks at zero extension. Scanning electron microscopy.-After the mechanical tests were conducted, specimens of interest were observed in the scanning electronmicroscope. A very careful dehydration procedure was used, the specimens being immersed in increasing strengths of acetone (50, 60, 70, 80, 90, 95, 100%, 20 min at each step) followed by critical-point drying using C02 as the drying liquid in a Polaron E 3000 critical-point drying apparatus. The dried specimens were cut and mounted in different orientations to show rupture and
structural details on marked i-inch aluminium stubs and coated with gold/palladium in a Polaron E 5000 sputter coating unit to produce a coating 150-200 A thick. The coated specimens were examined in a Cambridge Stereoscan S4-10 scanning electronmicroscope at an accelerating voltage of 20kV and filament current of 2-7 amps, the results permanently recorded with an Exacta %X5000 camera on Ilford FP4 135 roll film. Determination of calcium, coUagen and eiastin content.-The techniques used to determine the calcium, collagen and elastin contents of the rings after the mechanical tests were conducted are shown in Fig. 1. The calcium contents were expressed as ja/g dr-y wt of tissue, whilst the collagen and elastin contents were defined as % dry wt of the tissue samples analysed. RESULTS
Stress-strain curves of the aorta strips tested mechanicallv are shown in Fig. 2. Two factors appear to emerge from the observations of the effect of calcium ions on the mechanical properties of intact aorta media. First, there is an increase in the final modulus with only a slight decrease in the breaking stress and
3
Ca2+
_2_ x
E z 0 U)
0
40
60
Strain (%) Fig. 2.-Stress-strain behaviour of the rings from the thoracic
aortae of
pigs.
MECHA-NICAL BEHAVIOUR OF THE AORTA
strains and, secondlv, a decrease in the storage energy or area under the stressstrain curve. The calcium introduced into the elastin framework had the effect of stiffening the matrix of the wall but could not extend the network more than the GCGCG-treated rings. The storage energy, final moduli, breaking stress and
575
breaking strains of the 4 test svstems are summarized in the Table. The ultimate breaking stress along the length of the aorta increased for the intact aorta, and for rings treated with calcium it remained fairly constant (Fig. 7). The calcium content within the rings increased down the aorta up to 18 cm
Fig. 3.-Scanning electronmicrograph of the ruptured end of a control aortic ring; the internal surface is on the bottom right. x 60. Fig. 4.-Scanning electronmicrograph of the lamellae in the region of the rupture of a control aortic ring, showing some of the lamellae separating, exposing the inter-lamella structures. x 120. Fig. 5.-Scanning electronmicrograph of the ruptured end of an aortic ring treated with calcium. x 60.
Fig. 6.-Scanning electronmicrograph of the lamellae within an aortic ring treated with calciujm. x 120.
5w7 6
R. J. MINNS AND F. S. STEVEN
0 x
E z S S S
,
ca2+
S b. .0 C
ES2
a
0
5 10 15 L - ditauorg ta (ams) Fig. 7. Variation of the ultimate breaking stress (or maximum recorded load divided by the initial cross-section area) along the length of the pigs thoracic aorta. L = 0 being the top of the aortic arch.
from the thoracic arch, with an approximate twentv-fold increase in the values of the calcium-treated rings over the control but onlv double when calcium was introduced into the elastin-prepared rings. The values of the GCGCG-treated control rings before calcium treatment had apparent higher calcium content than the control values of the intact rings because the values were estimated for the tissue dry wt after treatment for the removal of the non-elastin components (Fig. 8). The dry wt elastin content fell, whilst the collagen content rose slightly along the length of the aorta, the total fibrous content (elastin and collagen) falling from 72 to 57%0 dry wt (Fig. 9). W7hen the ruptured rings were observed in the scanning electronmicroscope, the intact rings broke "cleanly" leaving an even fracture surface (Fig. 3) with little disruption of the lamellae (Fig. 4). Rings treated with calcium and ruptured exhibited a contrasting fracture surface, the surface breaking unevenly, leaving a "ragged" torn surface (Fig. 5), the individual lamellae appearing to be grossly disrupted (Fig. 6). The morphological findings from scanning electronmicroscopy are summarized in Fig. 10 which shows diagramatically the plan and internal views of the rupture phenomena.
200
150-
0
E 1 100
_
0
E 13 a
A
U
5oF AA ( 0
DOw
10 L - length aong Aorta (ams)
20
Fig. 8.-Variation of calcium content along the aorta. 0-Control, A-GCGCG-treated, A-GCGCG and Ca++, *-Ca++-treated intact rings.
577
MECHANICAL BEHAVIOU'R OF THE AORTA
40 .C
a.0
6 12 1 L - ngth along Aorta (cms) Fig.9. Varation of elastin and collagen content along the aorta. C-Elastin, 0-Collagen. O
TABLE.--Summary of Storage Energy, Final Modulus, Breaking Stress, and Breaking Strain of the Test Systems Breaking stress Treatment Control Ca++ GCGCG Ca-GCGCG -
(N/M2)
Breaking strain
(°h)
x 106
2 -916 : 0 -25 2-537 T 0-21 0-368 0-015
0-138 0: 0-012
62-0 2 2-1
60-3 2 2-0
43.4 = 1-2
46-4 = 1-0
DISCUSSION
It is clear from the analysis of the calcium content in the intact and elastinprepared media that the calcium introduced binds to the collagen framework and not to the elastic tissue. Collagen contains many carboxyl groups, and the positive calcium ions are bound by ionic interaction of aspartic and glutamic acid residues within the polypeptide chains of the polymeric collagen fibril to these sites. Elastin has very few carboxyl groups; thus there is little or no binding of calcium ions by ionic interaction, and even though considerable amounts of calcium ions were introduced into the
Stored energy (N/M2) X 104 48-9 - 4-1
31-25 2-7 3.3 2- 0-4 1-2 = 1-0
Final modulus (N/M2) X 106 15-0
28-0 3-0 1 -2
elastin framework there was little effect on the mechanical properties and only approximatelv double the amount of detectable calcium. In the intact media which contains collagen and elastin, the binding of calcium greatly increased by equilibrium with a low concentration of calcium chloride. The ionic binding of calcium must therefore be associated with the carboxyl and sulphate groups of the collagen fibrils and proteoglycans respectively. Although the ground substance plays a small role in the mechanical behaviour of the aorta and is particularly related to its time-dependent response (Minns et al., 1973), the collagen fibres are
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R. J. MIN"NS ANYD F. S. STEVEN
lagen content; however, when calcium is introduced, the breaking stress becomes lower than the intact aorta distallv. Clinically, lesions associated with calcium plaques and intimal thickening (Kadar et al., 1975) give rise to ruptures in the distal portion of the thoracic aorta, possibly related to the higher collagen content distally and hence the ability to bind more calcium, making the aorta wall more brittle and susceptible to rupture. Introduction of calcium has a dramatic effect on the morphology at the rupture site. In the normal intact aorta rings, the rupture was a clean break through the wall; the lamellae were all ruptured at the same level. The clean breaks are similar to those observed clinicallv in traumatic rupture of normal aortae (Sevitt, 1977); presumably the tensile strength of the collagen/elastin framework is exceeded at the same level through the wall. In the case of the calcium-treated aorta, the lamellae were very disrupted and the appearance of the rupture suggested b breakage at different levels and sites Fig. 10.-Sketches of the mode of failure of through the wall. The collagen/elastin the specimens. (a) The intact control rings broke cleanly through the wall from the framework appears to be structurallv inside, whilst (b) the calcium-treated specidisrupted similar to that observed within mens, although failing from the inside, showed gross disruption of the lamellae arteriosclerotic lesions (Kadar et al., 1975), during failure. the collagen fibrils being mechanicallv more brittle at sites where calcium is significantly related to the load and bound, producing the uneven rupture extension properties of the wall. appearance observed. The introduction of calcium within the We wish to thank Professor G. R. media had the effect of increasing the final modulus or stiffness (slope of the Higinson for the mechanical testing stress-strain curve) near the point of facilities, Dr G. Williams and ir D. S. rupture in both the elastin preparation and Page, Department of Pathology, Unithe intact specimen. In the calcium- versitv of Manchester for the scanning treated intact specimens the breaking electronmicroscope facilities and 3irs J. stress decreased. These two characteris- Watkins in the Department of Medical tics of increased stiffness and lowered Biochemistrv, Universitv of Manchester. ultimate tensile stress together with a We would also like to thank Mliss K. fall in storage energy suggest the media McConnon for conducting the calcium becomes "brittle", whilst in the elastin- analysis, and Dr E. G. Cleary for helpful prepared specimens the ultimate tensile discussions. stress doubled when calcium was introREFERENCES duced. The increase in breaking stress as M. (1973) Distensibility T. & HASEGAWA, -AzumA, one progresses distally along the aorta of the Vein: from the Architectural Point of may be reflected in the increase in colView. Biorheology, 10, 469.
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