- Email: [email protected]
Histologic, histochemical, and biomechanical properties of fragments isolated from the anterior wall of abdominal aortic aneurysms
BASIC RESEARCH STUDIES
Histologic, histochemical, and biomechanical properties of fragments isolated from the anterior wall of abdominal aortic aneurysms José Augusto Tavares Monteiro, MD, PhD,a Erasmo Simão da Silva, PhD,a Madhavan L. Raghavan, PhD,b Pedro Puech-Leão, PhD,a Maria de Lourdes Higuchi, PhD,a and José Pinhata Otoch, PhD,a São Paulo, Brazil; and Iowa City, Iowa Objective: To analyze biomechanical, histologic, and histochemical properties of anterior fragments of abdominal aortic aneurysms (AAA) and to correlate them with the maximum transverse diameter (MTD) and symptoms associated to the aneurysms. Methods: Fragments of the anterior aneurysm wall were obtained from 90 patients submitted to open repair of AAA of degenerative etiology from 2004 to 2009 in the Clinics Hospital of São Paulo University Medical School. Two specimens were produced from the fragments: one for histologic analysis for quantification of collagen fibers, elastic fibers, smooth muscle cells, and degree of inflammatory activity and the other for uniaxial tensile test to assess biomechanical failure properties of the material, such as strength, tension, and stress. Cases were classified according to symptoms and to the AAA MTD. Results: Fragments from AAA with MTD $ 5.5 cm showed higher values for biomechanical failure properties than those of AAA with MTD < 5.5 cm (strength, 5.32 6 2.07 3 4.1 6 2.41 N; tension, 13.83 6 5.58 3 10.82 6 6.48 N/cm; stress, 103.02 3 77.03 N/cm2; P < .05). No differences were observed between the groups in relation to failure strain (0.41 6 0.12 3 0.37 6 0.14; P [ .260) and thickness of the fragments (1.58 6 0.41 3 1.53 6 0.42 mm; P [ .662). The average values of fiber compositions of all the fragments were as follows: collagen fibers, 44.34 6 0.48% and 61.85 6 10.14% (Masson trichrome staining and Picrosirius red staining, respectively); smooth muscle cells, 3.46 6 2.23% (immunohistochemistry/alpha-actin); and elastic fibers, less than 1% (traces) (Verhoeff-van Gieson staining). No differences in fiber percentages (collagen, elastic, and smooth muscle) were observed in fragments from AAA with MTD $5.5 cm and <5.5 cm, but more intense inflammatory activity was seen in larger AAA (grade 3; 70% 3 28.6%; P [ .011). Compared with asymptomatic aneurysms, symptomatic aneurysms showed no differences in the biomechanical failure properties (strength, 5.32 6 2.36 3 4.65 6 2.05 N; P [ .155; tension, 14.08 6 6.11 3 12.81 6 5.77 N/cm; P [ .154; stress, 103.02 3 84.76 N/cm2; P [ .144), strain (0.38 6 0.12 3 0.41 6 0.13; P [ .287), thickness of the fragments (1.56 6 0.41 3 1.57 6 0.41 mm; P [ .848), and histologic composition (collagen fibers, 44.67 6 11.17 3 44.02 6 13.79%; P [ .808; smooth muscle fibers, 2.52 3 2.35%; P [ .751; elastic fibers, <1%) Conclusions: Fragments of the anterior wall from larger aneurysms were more resistant than those from smaller AAA, with no tissue properties that could explain this phenomenon in the histologic or histochemical analyses utilized. (J Vasc Surg 2014;59:1393-401.) Clinical Relevance: The fragments of the anterior midsection from larger aneurysms were more resistant than those from smaller abdominal aortic aneurysms, with no tissue properties that could explain this phenomenon in the histologic or histochemical analyses. Larger aneurysms, at least in this place may be stronger than smaller aneurysms. It could point toward regional differences (heterogeneity, localized pathologies) as an important player in aneurysm rupture. Uniaxial strain tests are an important tool for the comprehension of a complex behavior such as that from an aneurysmal aortic wall. However, these tests still have limitations in providing information that would allow the calculation of the risk of rupture for abdominal aortic aneurysms.
From the Department of Surgery, Vascular and Endovascular Division and Surgical Technique Division, University of São Paulo Medical School, São Pauloa; and the Department of Biomedical Engineering, University of Iowa, Iowa City.b Author conflict of interest: none. Presented as a free presentation at the Third International Meeting on Aortic Diseases held in Liege, Belgium, October 4-6, 2012. Additional material for this article may be found online at www.jvascsurg.org. Reprint requests: José Augusto Tavares Monteiro, MD, PhD, Department of Surgery, Vascular and Endovascular Division and Surgical Technique
Division, University of São Paulo Medical School, Avenida Dr Enéas de Carvalho Aguiar, 155, 6 andar, bloco 7, CEP 05403-000, São Paulo, SP, Brazil (e-mail: [email protected]). The editors and reviewers of this article have no relevant financial relationships to disclose per the JVS policy that requires reviewers to decline review of any manuscript for which they may have a conflict of interest. 0741-5214/$36.00 Copyright Ó 2014 by the Society for Vascular Surgery. http://dx.doi.org/10.1016/j.jvs.2013.04.064
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The most common parameter used to identify the risk of rupture for abdominal aortic aneurysm (AAA) is the maximum transverse diameter (MTD),1-3 and surgical repair is based on MTD greater than or equal to 5.5 cm.4-6 However, many AAA with MTD greater than 5.5 cm may not rupture7-9 and 10% to 24% of ruptured AAA have MTD of less than 5.5 cm.1,9,10 The risk of rupture may not, therefore, be based on a single criterion.11 Aortic dilation, progression, and rupture are considered a complex phenomena with important biomechanical component,12-14 justifying the measurements and behavior analysis of the biomechanical properties of the AAA wall as a tool for estimating the risk of rupture of a particular aneurysm.15 One possible approach is through testing of uniaxial stretching of aortic wall fragments associated with the study of the elements of structural composition of the tissue because they play different roles in the maintenance of elasticity, mechanical strength, and integrity of the wall.16-20 Studies using destructive uniaxial biomechanical tests with fragments of AAA in humans are few, with variable methodology, including analyzing fragments from cadavers and surgical patients even as different regions of aneurysms and elastic diagrams, with reduced sample, and with different conclusions (Table I).21-31 We proposed to resolve disputes by studying a larger number of specimens/individuals using specimens with less deterioration from individuals operated, standardizing the methodology of the tests, and adding the histologic analysis, seeking to correlate it to the biomechanical parameters found. Therefore, the objective of the present study is to analyze the parameters of biomechanical failure, thickness, as well as the amount of smooth muscle, elastic and collagen fibers, and inflammatory infiltration of the anterior longitudinal fragments of AAA removed from patients who underwent open surgical repair and to correlate them with the MTD and clinical manifestations of aneurysms. METHODS Design and eligibility. The protocol of this prospective histologic and mechanical study was approved by the Ethics Committee of Hospital das Clínicas, number 373/ 04. Participants were all consecutive patients with AAA of nonspecific, degenerative etiology, with indication for open repair from January 2004 to August 2009. During this period, 400 aneurysms were operated upon; however, only two surgeons from the group were involved with the study and 112 patients were initially selected. All subjects or relatives gave their informed consent for this study and the use of tissue specimens, together with the documentation for hospitalization and surgical procedures. The diameters of aneurysms were determined by computed tomography. The computed tomography apparatus was next to the emergency room, allowing patient examination without prejudice to the patient; there was no delay to the treatment. The presence of diabetes, hypertension, dyslipidemia, and coronary artery disease was made based on the previous history of the individual.
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Asymptomatic and symptomatic AAA (ie, abdominal pain, lumbar pain, pain during palpation of the AAA) and patients with ruptured AAA undergoing emergency surgery were included in the study. Any patient with pain unrelated to the dilation was considered asymptomatic, as were those who through image analysis, inventory cavity, and postoperative evolution were identified as having other causes of pain. Patients were not included in the study if the cause of AAA was not degenerative or if they had thoracic or thoracoabdominal aneurysms. They were excluded if the aneurysm was visually identified during surgery as inflammatory. For safety, we included as criteria for exclusion (1) bleeding or clinical instability during surgery; and (2) presence of any aneurysm feature that would prevent the collection of tissue samples suitable for the histologic and mechanical studies (eg, if the aneurysm was not large enough and the prosthesis could not be covered by the remaining aortic wall). There was no need to exclude patients from the study because of the causes stated above. All patients were operated in two hospital units of the same university campus (Instituto Central and Instituto do Coração, HCFMUSP) and by the same surgical team using the same prosthesis type (Dacron-knitted). Specimens. At the end of the surgery, a tissue fragment was removed from the remaining anterior wall of the AAA, the only possibility to be addressed without prejudice to the surgical technique dedicated to open AAA. This tissue fragment was removed so that its larger dimension would coincide with the greater axis of the vessel, longitudinally (Fig 1). The removed fragment was immediately immersed in saline and transferred to the Biomechanical Laboratory (Surgery Department), where it was preserved at 4 C. Each fragment was analyzed up to 48 hours after collection. From each tissue fragment collected during surgery, prior to histologic and biomechanical analysis, elements were removed outside the aneurysmal wall itself as mural thrombus, atheroma plaques, and retroperitoneal fat, through dissection of the aortic tissue. After that, two specimens were produced using a device designed specifically for this study composed of two parallel blades, each with an extension of 40 mm and a width of 5 mm. One was sent immediately to biomechanical test and the other was packaged immediately in a 5% buffered formalin and later embedded in paraffin for the histologic study (in the Pathology Laboratory in Instituto do Coração) identified with the same case number. We initially performed the biomechanical tests (until 2009). One of the authors was trained to read the slides. The author was blinded to the clinical characteristics, anatomic, and biomechanical study on the fragment. Mechanical test. One of the two aortic specimens of each patient was used in the mechanical test. An Instron In-Spec 2200 Benchtop Tester (Instron Corporation, Norwood, Mass) device with custom fabricated specimen bath and custom soft tissue grips was used (Fig 2, online only). Each specimen strip was clamped in the tissue grip attached to the crossheads of the test device. The length (L0), the width (W0), and the thickness (T0) of the
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Table I. Studies on the uniaxial tension of the wall of AAA First author
No.
Methods
Failure stress
Failure strain
Stiffness
He and Roach22
8 (3 from surgery 5 from autopsies)
Uniaxial tensile test; histologic analysis
-
-
Raghavan et al23
45
Uniaxial tensile test
\ Curve shifted to the left; slope was greater -
Thubrikar et al
5 from surgeries
Uniaxial tensile test
Vorp et al25
7 from surgeries
Uniaxial tensile test
ILT, 138 N/cm2; no ILT, 216 N/cm2
Raghavan et al26 Di Martino et al27
4 from autopsies 25 from surgeries
Uniaxial tensile test Uniaxial tensile test
Vande Geest et al28
34 from surgeries
Uniaxial tensile test
Vande Geest et al15 Xiong et al29 Raghavan et al30
38 from surgeries 14 from surgeries 11 from autopsies
Uniaxial tensile test Uniaxial tensile test Uniaxial tensile test Histology
Reeps et al31
50 from surgeries
Uniaxial tensile test
Present study
90 from surgeries
Uniaxial tensile test
126 N/cm2 (14 N/cm) Ruptured, 54 N/cm2 Elective, 82 N/cm2 87 N/cm2 67 N/cm2 80 N/cm2 93 N/cm2 AAA ruptured, 95 N/cm2 (11.2 N/cm) AAA unruptured, 98 N/cm2 (11.6 N/cm) 106.3 N/cm2 (15.23 N/cm) 103.14 N/cm2 (13.18 N/cm)
24
86 N/cm2 Z Range, 38-73 N/cm2
-
Range, The longitudinal 0.32-0.58 direction and posterior region were less stiff Wall weakening near the thick ILT 0.5 Less stiff AAA are weaker and prone to rupture 0.32 0.39
\
-
0.39
AAA, Abdominal aortic aneurysm; ILT, intraluminal thrombus.
specimen strip between the clamps were registered by the device and recorded for this study. The zero length was attained by placing a negligible preload on the specimen (0.01 N). Width and thickness were measured at three sites and averaged, and the arithmetic average of these considered the value of the specimen. Subsequently, the specimen bath was filled with saline at room temperature. Mechanical extension tests were performed under immersion in saline to maintain the moisture of the tissue (Fig 3, online only). Preconditioning was performed by loading and unloading the specimen to 5% of its length at 20% of specimen length/min aiming to promote the recruitment of fibers and reduce the hysteresis of the material. After preconditioning, the tissue strip was uniaxially extended also at 20% of specimen length/min until failure, while recording the force and extension at an acquisition rate of 1 Hz. The location of specimen failure with respect to the clamps was recorded to identify and discard specimens that failed too close to the clamp (within 2 mm) as this may have been because of tissue damage or slip during clamping. The traction device works under thrust from the water and as it unfolds during the biomechanical test and rises from the water; it, therefore, suffers less thrust (Fig 4, online only). Consequently, it was necessary to correct this influence by filling saline to the same level and subtracting the force extension data recorded without the specimen from the data for tests with each specimen. The correction factor for thrust was added to a calculation spreadsheet and used to
produce the final results considering the elastic diagram data (the set of points that correlate the specimen deformation with the strength applied to it during the biomechanical test). This protocol was first described by Raghavan et al in 1996.23 A specimen may be tested only once in the case of destructive uniaxial tensile testing. Only one researcher (J.M.) performed all mechanical tests. Mechanical test calculations. The force extension data from the mechanical testing were used to determine the peak load (Ff) and extension at peak load (Lf L0). The failure properties calculated were failure strain, failure tension, and failure stress, as follows.26 Failure strain (extension at peak load as a fraction of original length): Df ¼ L f L 0 L 0 Failure tension (peak load per unit width of specimen strip): pffiffiffi Ff W f ¼ Ff W 0 1 þ Df Failure stress (peak load per unit cross-sectional area of specimen strip): Ef ¼ Ff A f ¼ Ff W 0 T0 1 þ Df
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Fig 1. A, Aneurysmal wall remaining postinterposition of synthetic prosthesis. B, Removal of the fragment with the largest possible dimensions, being the greatest dimension coincident with the longitudinal axis of the aorta.
Where L0, W0, and T0 are the length, the width, and the thickness of the specimen strip between the clamps at zero load. The Ef term in the above equations account for the reduction in cross-sectional area in case of stress calculations and reduction of width for tension calculations. Histologic analysis. The second specimen from the same patient, contiguous from the first, was used for histologic analysis in the Pathology Laboratory of Instituto do Coração Hospital. It was preserved in formaldehyde, paraffin-embedded, and cut in 5-mm slices. The slides were prepared, always by the same laboratory technician, for analysis of the inflammatory activity and count of collagen, elastic, and smooth muscle fiber, stained with hematoxylin and eosin, Masson trichrome, Picrosirius, Verhoeff-van Gieson, and for immunohistochemistry using smooth muscle actin antibody (M0851; Dako Cytomation, Glostrup, Denmark) and brown diaminobenzidine chromogen. The same biologist/laboratory technician read all immunohistochemistry slides. The slides, with different dyes corresponding to one specimen, were marked with a line in three identical adjoining sites, transversally, through superposition. Next, the reading of each slide was performed in the field obtained with optical magnification five times to the left of the previously marked locations. Hematoxylin and eosin staining allowed inflammation intensity evaluation; the semiquantitative analysis considered 0 points for inflammation absence, 1 point for mild (small foci with few and scattered cells), 2 points for moderate (less foci and number of cells), or 3 intense (big foci or numerous scattered cells). One biologist made all inflammatory activity analyses. The image obtained by microscopy was captured by a 3CCD video camera Donpisha (model XC 003; Sony, Tokyo, Japan), coupled to the microscope, and transmitted to the computer with the program Image Analysis System from Leica (Quantimet-500; Leica Microsystems, Wetzlar, Germany). The percentage of the elements under study on the slide with their staining was obtained by manual demarcation of the element of interest and quantified by the software. The result for each element corresponds to the arithmetic mean of the percentage measured in each of three adjacent fields (by the same researcher, J. M. for all cases). Statistical analysis. Categorical variables are presented in tables with absolute (n) and relative (%) frequencies. The
association between them was evaluated with the c2, Fisher exact, or with the likelihood ratio tests. Quantitative variables were presented descriptively in tables containing means, standard deviations, or medians, and first and third quartiles. The means of parametric variables were evaluated with the Student t-test or with analysis of variance. When significant, we used the Tukey test to discriminate differences. The distributions of nonparametric variables were assessed with the Mann-Whitney or Kruskal-Wallis analysis. When these were significant, we used the Dunn test to discriminate differences. P values of <.05 were considered statistically significant.32 The hypothesis checked was that the existence of differences in resistance and/or in composition among fragments from aneurysms larger or equal to or smaller than 5.5 cm and between symptomatic and asymptomatic aneurysms. RESULTS During the study period, 112 patients operated with AAA in our service were included. However, five AAA were inflammatory and were excluded during surgery. Specimens collected from the remaining 107, allowing prosthesis implantation and adequate coverage, were considered. During mechanical tests, 17 cases were further excluded because specimens slipped or ruptured close to the device clamps (up to 2 mm far from the clamps). Therefore, this study was completed with 90 cases. Among the 90 patients, 66 (73.3%) were men, 74 (82.2%) had hypertension, 38 (42.2%) had dyslipidemia, 7 (7.77%) had diabetes, and 78 (86.66%) were active or exsmokers. Aneurysms were 3.5-12 cm in diameter (mean: 6.54 6 1.66 cm), and 45 patients (50%) were asymptomatic. Among the 45 patients with symptoms, 10 aneurysms were ruptured. Aneurysms were divided into two groups, those with a MTD smaller than 5.5 cm and those with a MTD equal to or greater than 5.5 cm, and the association between the size category and demographic or clinical characteristics of the patients was searched. Only age was significantly associated with MTD: individuals with smaller aneurysms were significantly younger (P ¼ .049). Sex and prevalence of diabetes, hypertension, coronary artery disease, dyslipidemia, tobacco use, and family antecedents for AAA were not different.
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Table II. Physical and biomechanical measurements and histologic and histochemical properties (mean 6 standard deviation) according to the MTD of AAA Variable Thickness, mm Mean diameter, cm Failure load, N Failure strain Failure stress, N/cm2 Failure tension, N/cm Collagen area % (Masson) Collagen area % (Picrosirius) % Alpha actin/vessel % Alpha actin/adventitia Elastin area % (Verhoeff-van Gieson)c
MTD < 5.5 cm (n ¼ 25)
MTD $ 5.5 cm (n ¼ 65)
P
1.53 6 0.42 4.89 6 0.53 4.1 6 2.41 0.37 6 0.14 77.03 (53.32-111.71) 10.82 6 6.48 45.32 6 13.73 62 (56-70.5) 3.17 (1.08-6.22) 3.04 (2.49-4.22) <1
1.58 6 0.41 7.28 6 1.45 5.32 6 2.07 0.41 6 0.12 103.02 (75.70-144.42) 13.83 6 5.58 43.97 6 12.06 61 (56-67) 1.99 (1.48-4.22) 2.74 (1.47-4.2) <1
.662a .019a .260a .027b .020a .648a .350b .361b .322b
AAA, Abdominal aortic aneurysm; MTD, maximum transverse diameter. a 2 c test. b Mann-Whitney test. c Less than 1% were considered trace elements.
Table III. Physical and biomechanical measurements and histologic and histochemical properties (mean 6 standard deviation) according to the presence of symptoms in patients with AAA Variable
Asymptomatic (n ¼ 45)
Ruptured and symptomatic (n ¼ 45)
P
Mean diameter, cm Thickness, mm Failure load, N Failure strain Failure stress, N/cm2 Failure tension, N/cm Collagen area % (Masson) Collagen area % (Picrosirius) % Alpha actin/vessel % Alpha actin/adventitia Elastin area % (Verhoeff-van Gieson)c
6.09 (5.30-6.75) 1.57 6 0.41 4.65 6 2.05 0.41 6 0.13 84.76 (64.08-121.05) 12.81 6 5.77 44.02 6 13.79 62.00 (56.00-69.50) 2.35 (1.52-5.86) 2.81 (1.54-3.50) <1
7.15 (5.70-8.60) 1.56 6 0.41 5.32 6 2.36 0.38 6 0.12 103.02 (75.63-147.99) 14.08 6 6.11 44.67 6 11.17 60.00 (55.00-65.50) 2.52 (1.14-5.91) 3.10 (2.18-5.77) <1
.009a .848b .155b .287b .144b .154b .808b .120a .751a .169a
AAA, Abdominal aortic aneurysm a Mann-Whitney test. b Student t-test. c Less than 1% were considered trace elements.
The results from the biomechanical tests of AAA fragments were compared between larger and smaller aneurysms, and, as shown in Table I, the mean values of failure load, failure stress, and failure tension were significantly higher for larger AAA specimens. No differences were found comparing larger and smaller aneurysms regarding thickness or failure strain. Table II shows also the results from the histologic and histochemical analyses, but again no significant differences were found between larger and smaller aneurysms. The same demographic and clinical variables were investigated in relation to symptoms. The only clinical variable significantly associated with asymptomatic aneurysms was dyslipidemia (P < .001). Sex, age, and prevalence of other clinical variables were not different. The mean diameter of aneurysms was significantly larger among patients with symptoms or ruptured AAA, as shown in Table III. No other significant difference was found in histologic, histochemical, or biomechanical
variables comparing symptomatic or asymptomatic patients. In search for a discriminative variable, another analysis was made joining symptoms and AAA diameter, as described in Table IV. Considering specimens of smaller aneurysms from asymptomatic patients, specimen resistance was significantly lower, as shown by the failure load and failure tension variables. However, again, no histochemical feature could discriminate the lesions. Only inflammatory activity was shown to be higher in AAA with a larger diameter (category 3 of inflammatory activity MTD < 5.5 cm 28.6% MTD > 5.5 cm 70%; P ¼ .011) but with no differences regarding the presence of symptoms. DISCUSSION The present study analyzed fragments of the anterior wall of AAA removed during corrective open surgery and is the largest case series, according to our knowledge22-31 of destructive uniaxial biomechanical tests analyzed by
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Table IV. Physical and biomechanical measurements and histologic and histochemical properties (mean 6 standard deviation) according to the presence of symptoms in patients with AAA and the MTD of aneurysms
Variable
Asymptomatic MTD < 5.5 cm (n ¼ 15)
Symptomatic MTD < 5.5 cm (n ¼ 10)
Asymptomatic MTD > 5.5 cm (n ¼ 30)
Symptomatic MTD > 5.5 cm (n ¼ 35)
P
Mean diameter, cm 5.21 6 0.56 4.84 6 0.50 6.67 6 0.86 7.68 6 1.51 Thickness, mm 1.39 6 0.3 1.75 6 0.49 1.66 6 0.44 1.5 6 0.37 Failure load, N 3.43 6 1.9 5.09 6 2.83 5.25 6 1.88 5.38 6 2.26 Failure strain 0.37 6 0.13 0.37 6 0.16 0.43 6 0.13 0.39 6 0.1 Failure stress, N/cm2 77.03 (34.82-102.77) 85.90 (57.05-129.13) 90.06 (70.38-130.99) 104.69 (82.58-151.89) Failure tension, N/cm 9.15 6 4.90 13.34 6 7.94 13.84 6 5.59 14.29 6 5.60 Collagen area % (Masson) 43.87 6 15.09 47.5 6 11.82 44.1 6 13.36 43.86 6 11.02 Collagen area % (Picrosirius) 62.00 (61.00-67.00) 56.00 (53.00-74.00) 61.00 (55.25-73.75) 60.00 (56.00-65.00) % Alpha actin/vessel 4.92 (1.32-6.22) 2.77 (1.00-7.50) 2.02 (1.50-3.17) 1.74 (1.27-5.91) % Alpha actin/adventitia 3.16 (2.23-3.55) 2.97 (2.44-7.56) 2.16 (1.46-3.06) 3.68 (1.64-5.55) Elastin area % (Verhoeff-van <1 <1 <1 <1 Gieson)e
.056a .029a,b .354a .097a,c .036a,d .871a .367a .702a .306a
AAA, Abdominal aortic aneurysm; MTD, maximum transverse diameter. a Kruskal-Wallis test. b Specimens from asymptomatic patients with AAA smaller than 5.5 cm had significantly lower values than asymptomatic patients with greater lesions and symptomatic patients with greater lesions. c Specimens from asymptomatic patients with AAA smaller than 5.5 cm had significantly lower values than symptomatic patients with greater lesions. d Specimens from asymptomatic patients with AAA smaller than 5.5 cm had significantly lower values than symptomatic patients with greater lesions. e Less than 1% were considered trace elements.
a clinical, morphologic, and histologic point of view; data obtained from 90 cases were considered appropriate for all evaluations. The use of methods consecrated by Raghavan23 in 1996 and the correlation of these results with those of a previous study conducted by the same group, give consistency to the findings (Table V).33,34 For a better comprehension of our line of reasoning, we chose four different cases in our sample, notwithstanding isolated patients, to reflect the behavior of the groups to which they belong (from larger and smaller AAA, symptomatic or asymptomatic) and also chose one tissue sample from a normal cadaveric abdominal aorta (without aneurysm) from another study in progress conducted by the same group in accordance with values reported for biomechanical properties of nonaneurysmal aorta in a previous study involving 26 individual cadavers (Table V).34 We used the same methodology to study normal cadaveric aortas and aneurysmal segments of surgical patients. The specimen relative to normal aorta was obtained from a cadaver because it would not be possible to obtain it from patients undergoing surgery, unlike the specimens regarding aneurysmal aorta. With these, we included Fig 5 with the respective elastics diagrams. As shown in Fig 5 and Table IV, as we moved from the group of small and asymptomatic aneurysms to the group of large and symptomatic aneurysms, we observed an increase in the maximum failure strength and failure tension of fragments associated with similar values of failure strain characterizing greater stiffness. As they increased in diameter, corresponding specimens were stiffer and more resistant; possibly as the aneurysm grows, the wall adapts, giving the tissue conditions to withstand larger loads. In this study, it seems that the MTD was more of a determinant of the biomechanical characteristics of the AAA than the patients’
Table V. Table comparing values found in this study for anatomic, biomechanical, and histologic parameters for fragments of the anterior wall of AAA and values obtained in other studies conducted by the same group for fragments of the anterior wall of corpses and AAA and ANA from autopsy
Age Diameter, cm Thickness, mm Failure strain Failure stress, N/cm2 Failure tension, N/cm Collagen % Masson Elastin % Verhoeff Smooth muscle fibers %
AAA surgery (n ¼ 90)
AAA autopsy (n ¼ 13)
69 6.62 1.57 0.39 103.1 13.1 44.37/61.91 <1 4.9
74 5.25 1.60 0.38 96.8 11.4 55.2 4.6 7.2
ANA autopsy (n ¼ 26) 62 1.42 0.47 137.2 14.7 49.3 17.1 30.2
AAA, Abdominal aortic aneurysm; ANA, normal aorta.
symptoms. Although behaviors were distinct from each other, specimen from AAA showed similar behavior compared with the specimen from the normal aorta: lower failure strain, lower resistance, and greater stiffness. These behaviors can be explained, at least in part, by our findings on histologic analysis. We observed also that the percent of elastic and smooth muscle fibers were both very reduced: less than 1% and around 2.5%, respectively, in small and large aneurysms. This could be explained by the fact that degradation of elastic and smooth muscle fibers happen very early in the course of aneurysmatic disease, with the steepest decline in the first stages.20 After the first aneurysmatic dilatation, structural or conformational change of the wall would influence the
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Fig 5. Elastic diagram of individual specimens of abdominal aortic aneurysms (AAA) obtained in vivo and normal aorta from a cadaver: load (N) in the vertical axis and distension (mm) in the horizontal axis. The purple, green, yellow, and red lines are from specimens from larger or smaller individual AAA used in this study from asymptomatic (AST) or symptomatic (ST) patients. The blue line shows data from the same biomechanical test with tissue from normal, not aneurysmatic aorta from a cadaver.
acting forces of flow and hemodynamics with more radial and circumferential stress,35 leading to progressive expansion and loss of integrity36,37; remodeling would be an important physiological response to these changes.35 In aneurysms of different sizes, an enlargement of the arterial wall volume (ie, an increase in the diameter and surface with preservation of the thickness) and preservation of the percent of collagen fibers reveal an increase in the absolute mass of collagen, supporting the thought that the expansion of the AAA is followed by the wall remodeling. The concurrent increase in the arterial wall volume, maintenance of collagen fibers percent, and reduction in the elastic and smooth muscle fibers in AAA compared with normal tissue inspires the hypothesis that other cellular components (not studied here), such as the extracellular amorphous matrix, the quality of reposition material, and the final structure, should influence the function of the tissue. Fig 5 shows the behavior of the asymptomatic and small aneurysm (red line), which already has a reduced count of elastic and smooth muscle fibers and a reduced thickness of the wall, reflecting lower degree of remodeling and, consequently, less resistance to force and distension. This is consistent with the idea of elasticity loss as an early event after the first aneurysmatic dilatation. The small, symptomatic AAA (green line in the Fig 5) can be exposed to more stimuli, influencing remodeling and thickness growth earlier at the presence of symptoms. Cellular infiltrate count in AAA wall evidenced that larger aneurysms had an increased inflammatory activity in this study. However, even using a larger sample, this study
could not find a statistically significant association between inflammatory activity and the presence of symptoms, although there were a larger percentage of asymptomatic cases in category 3 of inflammation. Possibly, inflammation is not a marker for AAA rupture, but a stimulus to wall remodeling. Certain types of cells composing the AAA wall would be more or less responsive to mechanical stimuli, changing the composition of the wall tissue,38 and this could also happen regarding inflammatory activity, as a marker of reinforcement and not deterioration of the wall. The fact that larger aneurysms are more likely to rupture, as shown in other studies,1-3 does not mean that they have fragile walls compared with aneurysms with smaller diameter. The remodeling response has been shown to be heterogeneous even in studies by the same research team,26,30 with wide intervals for the biomechanical variables in different sites of the same aneurysms and in different aneurysms. Besides, the rupture event is one that arises from the interaction of two components: wall strength and the stress generated on it by external elements. An aneurysm of greater diameter, that theoretically shows a more resistant wall, could be exposed to more intense stress.11,39 Aneurysms with less resistant walls can be exposed to forces and stresses smaller and are, thus, less prone to breakage. One limitation of the present study is the use of fragments just from the anterior wall of AAA. Tissue remodeling is a heterogeneous process, with different regions of the same aneurysm showing different resistances40 in a manner that biomechanical data from one wall facet and
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extrapolated to all facets of the aorta are limited in offering tools for rupture risk calculation. However, in study with operated patients, the use of posterior, lateral, and anterior faces of the wall is not possible, once we must close the remaining aortic wall over the prosthesis to obtain proper treatment.15 However, the site for specimen collection was standardized in this study for all cases.9,27 Additionally, the identification of the site of rupture because of the hematoma and the need for rapid intervention did not allow dissection to locate this new site. This fact would require a longer intervention in a critical patient. The removal of the anterior fragment takes a short time. Another limitation to consider is the use of uniaxial mechanical test; in vivo, the aneurysmatic wall is submitted to multiaxial loads, which lead to different stress situations.9,41,42 It is necessary to consider the complexity of the AAA wall. We are still searching for mathematical models to predict the biomechanical behavior of AAA. We have to feed the models with stress and distension test data to determine which uniaxial tests are viable as a standardized tool. In line with this, our study is the largest essay on the biomechanical and histologic properties of tissue from aneurysms collected in vivo, and we believe it helps to understand more of the physiology of such a complex structure as that of the AAA. CONCLUSIONS The fragments of the anterior midsection from larger aneurysms were more resistant to rupture than those from smaller AAA, with no tissue properties that could explain this phenomenon in the histologic or histochemical analyses. Larger aneurysms, at least in this anterior wall, may be stronger than smaller aneuryms. It could point toward regional differences (heterogeneity, localized pathologies) as an important player in aneurysm rupture. Uniaxial strain tests are an important tool for the comprehension of a complex behavior such as that from an aneurysmal aortic wall. However, these tests still have limitations in providing information that would allow the calculation of the risk of rupture of AAA. AUTHOR CONTRIBUTIONS Conception and design: JM, ES, MR, PP-L, MH, JO Analysis and interpretation: JM, ES, MR, PP-L, MH Data collection: JM, ES, MR, PP-L Writing the article: JM, ES, MR, PP-L, MH Critical revision of the article: JM, ES, MR, PP-L, MH, JO Final approval of the article: JM, ES, MR, PP-L, MH, JO Statistical analysis: JM, ES, MR Obtained funding: JM, ES, JO Overall responsibility: JM REFERENCES 1. Heng MS, Fagan MJ, Collier JW, Desai G, McCollum PT, Chetter IC. Peak wall stress measurement in elective and acute abdominal aortic aneurysms. J Vasc Surg 2008;47:17-22; discussion: 22.
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2. Wilson K, Bradbury A, Whyman M, Hoskins P, Lee A, Fowkes G, et al. Relationship between abdominal aortic aneurysm wall compliance and clinical outcome: a preliminary analysis. Eur J Vasc Endovasc Surg 1998;15:472-7. 3. Wilson K, Whyman M, Hoskins P, Lee AJ, Bradbury AW, Fowkes FG, et al. The relationship between abdominal aortic aneurysm wall compliance, maximum diameter and growth rate. Cardiovasc Surg 1999;7:208-13. 4. Mortality results for randomised controlled trial of early elective surgery or ultrasonographic surveillance for small abdominal aortic aneurysms. The UK Small Aneurysm Trial Participants. Lancet 1998;352:1649-55. 5. Lederle FA, Wilson SE, Johnson GR, Reinke DB, Littooy FN, Acher CW, et al. Immediate repair compared with surveillance of small abdominal aortic aneurysms. N Engl J Med 2002;346: 1437-44. 6. Brewster DC, Cronenwett JL, Hallet JW Jr, Johnston KW, Krupski WC, Matsumura JS. Joint Council of the American Association for Vascular Surgery and Society for Vascular Surgery. Guidelines for the treatment of abdominal aortic aneurysms. Report of a subcommittee of the Joint Council of the American Association for Vascular Surgery and Society for Vascular Surgery. J Vasc Surg 2003;37: 1106-17. 7. Simão da Silva E, Rodrigues AJ, Magalhães Castro de Tolosa E, Rodrigues CJ, Villas Boas do Prado G, Nakamoto JC. Morphology and diameter of infrarenal aortic aneurysms: a prospective autopsy study. Cardiovasc Surg 2000;8:526-32. 8. Darling RC, Messina CR, Brewster DC, Ottinger LW. Autopsy study of unoperated abdominal aortic aneurysm: the case for early resection. Circulation 1977;56(3 Suppl):II161-4. 9. Vorp DA. Biomechanics of abdominal aortic aneurysm. J Biomech 2007;40:1887-902. 10. Truijers M, Pol JA, SchultzeKool LJ, van Sterkenburg SM, Fillinger MF, Blankensteijn JD. Wall stress analysis in small asymptomatic, symptomatic and ruptured abdominal aortic aneurysms. Eur J Vasc Endovasc Surg 2007;33:401-7. 11. Vorp DA, Vande Geest JP. Biomechanical determinants of abdominal aortic aneurysm rupture. Arterioscler Thromb Vasc Biol 2005;25: 1558-66. 12. Tilson MD, Newman K. Rationale for molecular approaches to the etiology of abdominal aortic aneurysm disease. J Vas Surg 1992;15: 924-5. 13. Rasmussen TE, Hallet JW Jr, Tazelaar HD, Miller VM, Schulte S, O’Fallon WM, et al. Human leukocyte antigen class II immune response genes, female gender, and cigarette smoking as risk and modulating factors in abdominal aortic aneurysms. J Vasc Surg 2002;35:988-93. 14. Muluk SC, Gertler JP, Brewster DC, Cambria RP, LaMuraglia GM, Moncure AC, et al. Presentation and patterns of aortic aneurysms in young patients. J Vasc Surg 1994;20:880-6; discussion: 887-8. 15. Vande Geest JP, Wang DH, Wisniewski SR, Makaroun MS, Vorp DA. Towards a noninvasive method for determination of patient-specific wall strength distribution in abdominal aortic aneurysms. Ann Biomed Eng 2006;34:1098-106. 16. Di Martino E, Mantero S, Inzoli F, Melissano G, Astore D, Chiesa R, et al. Biomechanics of abdominal aortic aneurysm in the presence of endoluminal thrombus: experimental characterization and structural static computational analysis. Eur J Vasc Endovasc Surg 1998;15: 290-9. 17. Carmo M, Colombo L, Bruno A, Corsi FR, Roncoroni L, Cuttin MS, et al. Alteration of elastin, collagen and their cross-links in abdominal aortic aneurysms. Eur J Vasc Endovasc Surg 2002;23:543-9. 18. Watton PN, Hill NA. Evolving mechanical properties of a model of abdominal aortic aneurysm. Biomech Model Mechanobiol 2009;8: 25-42. 19. Jacob MP, Badier-Commander C, Fontaine V, Benazzoug Y, Feldman L, Michel JB. Extracellular matrix remodeling in the vascular wall. Pathol Biol (Paris) 2001;49:326-32. 20. Sakalihasan N, Heyeres A, Nusgens BV, Limet R, Lapière CM. Modifications of the extracellular matrix of aneurysmal abdominal aortas as a function of their size. Eur J Vasc Surg 1993;7:633-7.
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21. Mohan D, Melvin JW. Failure properties of passive human aortic tissue. I‒uniaxial tension tests. J Biomech 1982;15:887-902. 22. He CM, Roach MR. The composition and mechanical properties of abdominal aortic aneurysm. J Vasc Surg 1994;20:6-13. 23. Raghavan ML, Webster MW, Vorp DA. Ex vivo biomechanical behavior of abdominal aortic aneurysm: assessment using a new mathematical model. Ann Biomed Eng 1996;24:573-82. 24. Thubrikar MJ, Labrosse M, Robicsek F, Al-Soudi J, Fowler B. Mechanical properties of abdominal aortic aneurysm wall. J Med Eng Technol 2001;25:133-42. 25. Vorp DA, Lee PC, Wang DH, Makaroun MS, Nemoto EM, Ogawa S. Association of intraluminal thrombus in abdominal aortic aneurysm with local hypoxia and wall weakening. J Vasc Surg 2001;34:291-2. 26. Raghavan ML, Kratzberg J, Castro de Tolosa EM, Hanaoka MM, Walker R, da Silva ES. Regional distribution of wall thickness and failure properties of human abdominal aortic aneurysm. J Biomech 2006;39:3010-6. 27. Di Martino ES, Bohra A, Vande Geest JP, Gupta N, Makaroun MS, Vorp DA. Biomechanical properties of ruptured versus electively repaired abdominal aortic aneurysm wall tissue. J Vasc Surg 2006;43: 570-6; discussion: 576. 28. Vande Geest JP, Dillavou ED, Di Martino ES, Oberdier M, Bohra A, Makaroun MS, et al. Gender-related differences in the tensile strength of abdominal aortic aneurysm. Ann N Y Acad Sci 2006;1085:400-2. 29. Xiong J, Wang SM, Zhou W, Wu JG. Measurement and analysis of ultimate mechanical properties, stress-strain curve fit, and elastic modulus formula of human abdominal aortic aneurysm and nonaneurysmal abdominal aorta. J Vasc Surg 2008;48:189-95. 30. Raghavan ML, Hanaoka MM, Kratzberg JA, de Lourdes Higuchi M, da Silva ES. Biomechanical failure properties and microstructural content of ruptured and unruptured abdominal aortic aneurysm. J Biomech 2011;44:2501-7. 31. Reeps C, Maier A, Pelisek J, Härtl F, Grabher-Meier V, Wall WA, et al. Measuring and modeling patient-specific distributions of material properties in abdominal aortic aneurysm wall. Biomech Model Mechanobiol 2013;12:717-33. 32. Rosner B. Fundamentals of biostatistics. 4th ed. New York: Duxbury Press; 1994.
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33. Raghavan ML, Ikeda MH, Silva ES. Failure strength of abdominal aortic aneurysm: a necropsy study. New Orleans, LA: Proceedings of the ASME International Mechanical Engineering Congress and Exposition. Advances in Bioengineering; November 17-22 2002. 34. da Silva ES, et al. Oral Presentation. Sotckholm, Sweden: Proceedings of the Endovascular Surgery-Bringing Basic Science into Clinical Practice. Session 1-Basic Mechanism: Biomechanical aspects of abdominal and thoracic aorta according to the age; March 19-21, 2009. 35. Astrand H, Rydén-Ahlgren A, Sandgren T, Länne T. Age-related increase in wall stress of the human abdominal aorta: an vivo study. J Vasc Surg 2005;42:926-31. 36. Speelman L, Bosboom EM, Schurink GW, Hellenthal FA, Buth J, Breeuwer M, et al. Patient-specific AAA wall stress analysis: 99-percentile versus peak stress. Eur J Vasc Endovasc Surg 2008;36: 668-76. 37. Salsac AV, Sparks SR, Lasheras JC. Hemodynamic changes occurring during the progressive enlargement of abdominal aortic aneurysms. Ann Vasc Surg 2004;18:14-21. 38. Humphrey JD, Na S. Elastodynamics and arterial wall stress. Ann Biomed Eng 2002;30:509-23. 39. Di Martino ES, Guadagni G, Fumero A, Ballerini G, Spirito R, Biglioli P, et al. Fluid-structure interaction within realistic threedimensional models of the aneurysmatic aorta as a guidance to assess the risk of rupture of the aneurysm. Med Eng Phys 2001;23:647-55. 40. De Martino RR, Nolan BW, Goodney PP, Chang CK, Schanzer A, Cambria R, et al. Outcomes of symptomatic abdominal aortic aneurysm repair. J Vasc Surg 2010;52:5-12.e1. 41. Volokh KY. Comparison of biomechanical failure criteria for abdominal aortic aneurysm. J Biomech 2010;43:2032-4. 42. Vande Geest JP, Sacks MS, Vorp DA. The effects of aneurysm on the biaxial mechanical behavior of human abdominal aorta. J Biomech 2006;39:1324-34.
Submitted Dec 11, 2012; accepted Apr 29, 2013.
Additional material for this article may be found online at www.jvascsurg.org.
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Fig 2 (online only). A, Traction device Instron 2200 associated with Palm Top with Inspec software for control movements of the traction device. B, Laptop with Series IX software for data management.
Fig 3 (online only). Performing the test over immersion to preserve the moisture conditions.
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Fig 4 (online only). As occurred the motion of head traction, it emerges from the tub, thus reducing the volume of liquid displaced by it and consequently reducing the thrust that acted on it.
Histologic, histochemical, and biomechanical properties of fragments isolated from the anterior wall of abdominal aortic aneurysms José Augusto Tavares Monteiro, MD, PhD,a Erasmo Simão da Silva, PhD,a Madhavan L. Raghavan, PhD,b Pedro Puech-Leão, PhD,a Maria de Lourdes Higuchi, PhD,a and José Pinhata Otoch, PhD,a São Paulo, Brazil; and Iowa City, Iowa Objective: To analyze biomechanical, histologic, and histochemical properties of anterior fragments of abdominal aortic aneurysms (AAA) and to correlate them with the maximum transverse diameter (MTD) and symptoms associated to the aneurysms. Methods: Fragments of the anterior aneurysm wall were obtained from 90 patients submitted to open repair of AAA of degenerative etiology from 2004 to 2009 in the Clinics Hospital of São Paulo University Medical School. Two specimens were produced from the fragments: one for histologic analysis for quantification of collagen fibers, elastic fibers, smooth muscle cells, and degree of inflammatory activity and the other for uniaxial tensile test to assess biomechanical failure properties of the material, such as strength, tension, and stress. Cases were classified according to symptoms and to the AAA MTD. Results: Fragments from AAA with MTD $ 5.5 cm showed higher values for biomechanical failure properties than those of AAA with MTD < 5.5 cm (strength, 5.32 6 2.07 3 4.1 6 2.41 N; tension, 13.83 6 5.58 3 10.82 6 6.48 N/cm; stress, 103.02 3 77.03 N/cm2; P < .05). No differences were observed between the groups in relation to failure strain (0.41 6 0.12 3 0.37 6 0.14; P [ .260) and thickness of the fragments (1.58 6 0.41 3 1.53 6 0.42 mm; P [ .662). The average values of fiber compositions of all the fragments were as follows: collagen fibers, 44.34 6 0.48% and 61.85 6 10.14% (Masson trichrome staining and Picrosirius red staining, respectively); smooth muscle cells, 3.46 6 2.23% (immunohistochemistry/alpha-actin); and elastic fibers, less than 1% (traces) (Verhoeff-van Gieson staining). No differences in fiber percentages (collagen, elastic, and smooth muscle) were observed in fragments from AAA with MTD $5.5 cm and <5.5 cm, but more intense inflammatory activity was seen in larger AAA (grade 3; 70% 3 28.6%; P [ .011). Compared with asymptomatic aneurysms, symptomatic aneurysms showed no differences in the biomechanical failure properties (strength, 5.32 6 2.36 3 4.65 6 2.05 N; P [ .155; tension, 14.08 6 6.11 3 12.81 6 5.77 N/cm; P [ .154; stress, 103.02 3 84.76 N/cm2; P [ .144), strain (0.38 6 0.12 3 0.41 6 0.13; P [ .287), thickness of the fragments (1.56 6 0.41 3 1.57 6 0.41 mm; P [ .848), and histologic composition (collagen fibers, 44.67 6 11.17 3 44.02 6 13.79%; P [ .808; smooth muscle fibers, 2.52 3 2.35%; P [ .751; elastic fibers, <1%) Conclusions: Fragments of the anterior wall from larger aneurysms were more resistant than those from smaller AAA, with no tissue properties that could explain this phenomenon in the histologic or histochemical analyses utilized. (J Vasc Surg 2014;59:1393-401.) Clinical Relevance: The fragments of the anterior midsection from larger aneurysms were more resistant than those from smaller abdominal aortic aneurysms, with no tissue properties that could explain this phenomenon in the histologic or histochemical analyses. Larger aneurysms, at least in this place may be stronger than smaller aneurysms. It could point toward regional differences (heterogeneity, localized pathologies) as an important player in aneurysm rupture. Uniaxial strain tests are an important tool for the comprehension of a complex behavior such as that from an aneurysmal aortic wall. However, these tests still have limitations in providing information that would allow the calculation of the risk of rupture for abdominal aortic aneurysms.
From the Department of Surgery, Vascular and Endovascular Division and Surgical Technique Division, University of São Paulo Medical School, São Pauloa; and the Department of Biomedical Engineering, University of Iowa, Iowa City.b Author conflict of interest: none. Presented as a free presentation at the Third International Meeting on Aortic Diseases held in Liege, Belgium, October 4-6, 2012. Additional material for this article may be found online at www.jvascsurg.org. Reprint requests: José Augusto Tavares Monteiro, MD, PhD, Department of Surgery, Vascular and Endovascular Division and Surgical Technique
Division, University of São Paulo Medical School, Avenida Dr Enéas de Carvalho Aguiar, 155, 6 andar, bloco 7, CEP 05403-000, São Paulo, SP, Brazil (e-mail: [email protected]). The editors and reviewers of this article have no relevant financial relationships to disclose per the JVS policy that requires reviewers to decline review of any manuscript for which they may have a conflict of interest. 0741-5214/$36.00 Copyright Ó 2014 by the Society for Vascular Surgery. http://dx.doi.org/10.1016/j.jvs.2013.04.064
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The most common parameter used to identify the risk of rupture for abdominal aortic aneurysm (AAA) is the maximum transverse diameter (MTD),1-3 and surgical repair is based on MTD greater than or equal to 5.5 cm.4-6 However, many AAA with MTD greater than 5.5 cm may not rupture7-9 and 10% to 24% of ruptured AAA have MTD of less than 5.5 cm.1,9,10 The risk of rupture may not, therefore, be based on a single criterion.11 Aortic dilation, progression, and rupture are considered a complex phenomena with important biomechanical component,12-14 justifying the measurements and behavior analysis of the biomechanical properties of the AAA wall as a tool for estimating the risk of rupture of a particular aneurysm.15 One possible approach is through testing of uniaxial stretching of aortic wall fragments associated with the study of the elements of structural composition of the tissue because they play different roles in the maintenance of elasticity, mechanical strength, and integrity of the wall.16-20 Studies using destructive uniaxial biomechanical tests with fragments of AAA in humans are few, with variable methodology, including analyzing fragments from cadavers and surgical patients even as different regions of aneurysms and elastic diagrams, with reduced sample, and with different conclusions (Table I).21-31 We proposed to resolve disputes by studying a larger number of specimens/individuals using specimens with less deterioration from individuals operated, standardizing the methodology of the tests, and adding the histologic analysis, seeking to correlate it to the biomechanical parameters found. Therefore, the objective of the present study is to analyze the parameters of biomechanical failure, thickness, as well as the amount of smooth muscle, elastic and collagen fibers, and inflammatory infiltration of the anterior longitudinal fragments of AAA removed from patients who underwent open surgical repair and to correlate them with the MTD and clinical manifestations of aneurysms. METHODS Design and eligibility. The protocol of this prospective histologic and mechanical study was approved by the Ethics Committee of Hospital das Clínicas, number 373/ 04. Participants were all consecutive patients with AAA of nonspecific, degenerative etiology, with indication for open repair from January 2004 to August 2009. During this period, 400 aneurysms were operated upon; however, only two surgeons from the group were involved with the study and 112 patients were initially selected. All subjects or relatives gave their informed consent for this study and the use of tissue specimens, together with the documentation for hospitalization and surgical procedures. The diameters of aneurysms were determined by computed tomography. The computed tomography apparatus was next to the emergency room, allowing patient examination without prejudice to the patient; there was no delay to the treatment. The presence of diabetes, hypertension, dyslipidemia, and coronary artery disease was made based on the previous history of the individual.
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Asymptomatic and symptomatic AAA (ie, abdominal pain, lumbar pain, pain during palpation of the AAA) and patients with ruptured AAA undergoing emergency surgery were included in the study. Any patient with pain unrelated to the dilation was considered asymptomatic, as were those who through image analysis, inventory cavity, and postoperative evolution were identified as having other causes of pain. Patients were not included in the study if the cause of AAA was not degenerative or if they had thoracic or thoracoabdominal aneurysms. They were excluded if the aneurysm was visually identified during surgery as inflammatory. For safety, we included as criteria for exclusion (1) bleeding or clinical instability during surgery; and (2) presence of any aneurysm feature that would prevent the collection of tissue samples suitable for the histologic and mechanical studies (eg, if the aneurysm was not large enough and the prosthesis could not be covered by the remaining aortic wall). There was no need to exclude patients from the study because of the causes stated above. All patients were operated in two hospital units of the same university campus (Instituto Central and Instituto do Coração, HCFMUSP) and by the same surgical team using the same prosthesis type (Dacron-knitted). Specimens. At the end of the surgery, a tissue fragment was removed from the remaining anterior wall of the AAA, the only possibility to be addressed without prejudice to the surgical technique dedicated to open AAA. This tissue fragment was removed so that its larger dimension would coincide with the greater axis of the vessel, longitudinally (Fig 1). The removed fragment was immediately immersed in saline and transferred to the Biomechanical Laboratory (Surgery Department), where it was preserved at 4 C. Each fragment was analyzed up to 48 hours after collection. From each tissue fragment collected during surgery, prior to histologic and biomechanical analysis, elements were removed outside the aneurysmal wall itself as mural thrombus, atheroma plaques, and retroperitoneal fat, through dissection of the aortic tissue. After that, two specimens were produced using a device designed specifically for this study composed of two parallel blades, each with an extension of 40 mm and a width of 5 mm. One was sent immediately to biomechanical test and the other was packaged immediately in a 5% buffered formalin and later embedded in paraffin for the histologic study (in the Pathology Laboratory in Instituto do Coração) identified with the same case number. We initially performed the biomechanical tests (until 2009). One of the authors was trained to read the slides. The author was blinded to the clinical characteristics, anatomic, and biomechanical study on the fragment. Mechanical test. One of the two aortic specimens of each patient was used in the mechanical test. An Instron In-Spec 2200 Benchtop Tester (Instron Corporation, Norwood, Mass) device with custom fabricated specimen bath and custom soft tissue grips was used (Fig 2, online only). Each specimen strip was clamped in the tissue grip attached to the crossheads of the test device. The length (L0), the width (W0), and the thickness (T0) of the
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Table I. Studies on the uniaxial tension of the wall of AAA First author
No.
Methods
Failure stress
Failure strain
Stiffness
He and Roach22
8 (3 from surgery 5 from autopsies)
Uniaxial tensile test; histologic analysis
-
-
Raghavan et al23
45
Uniaxial tensile test
\ Curve shifted to the left; slope was greater -
Thubrikar et al
5 from surgeries
Uniaxial tensile test
Vorp et al25
7 from surgeries
Uniaxial tensile test
ILT, 138 N/cm2; no ILT, 216 N/cm2
Raghavan et al26 Di Martino et al27
4 from autopsies 25 from surgeries
Uniaxial tensile test Uniaxial tensile test
Vande Geest et al28
34 from surgeries
Uniaxial tensile test
Vande Geest et al15 Xiong et al29 Raghavan et al30
38 from surgeries 14 from surgeries 11 from autopsies
Uniaxial tensile test Uniaxial tensile test Uniaxial tensile test Histology
Reeps et al31
50 from surgeries
Uniaxial tensile test
Present study
90 from surgeries
Uniaxial tensile test
126 N/cm2 (14 N/cm) Ruptured, 54 N/cm2 Elective, 82 N/cm2 87 N/cm2 67 N/cm2 80 N/cm2 93 N/cm2 AAA ruptured, 95 N/cm2 (11.2 N/cm) AAA unruptured, 98 N/cm2 (11.6 N/cm) 106.3 N/cm2 (15.23 N/cm) 103.14 N/cm2 (13.18 N/cm)
24
86 N/cm2 Z Range, 38-73 N/cm2
-
Range, The longitudinal 0.32-0.58 direction and posterior region were less stiff Wall weakening near the thick ILT 0.5 Less stiff AAA are weaker and prone to rupture 0.32 0.39
\
-
0.39
AAA, Abdominal aortic aneurysm; ILT, intraluminal thrombus.
specimen strip between the clamps were registered by the device and recorded for this study. The zero length was attained by placing a negligible preload on the specimen (0.01 N). Width and thickness were measured at three sites and averaged, and the arithmetic average of these considered the value of the specimen. Subsequently, the specimen bath was filled with saline at room temperature. Mechanical extension tests were performed under immersion in saline to maintain the moisture of the tissue (Fig 3, online only). Preconditioning was performed by loading and unloading the specimen to 5% of its length at 20% of specimen length/min aiming to promote the recruitment of fibers and reduce the hysteresis of the material. After preconditioning, the tissue strip was uniaxially extended also at 20% of specimen length/min until failure, while recording the force and extension at an acquisition rate of 1 Hz. The location of specimen failure with respect to the clamps was recorded to identify and discard specimens that failed too close to the clamp (within 2 mm) as this may have been because of tissue damage or slip during clamping. The traction device works under thrust from the water and as it unfolds during the biomechanical test and rises from the water; it, therefore, suffers less thrust (Fig 4, online only). Consequently, it was necessary to correct this influence by filling saline to the same level and subtracting the force extension data recorded without the specimen from the data for tests with each specimen. The correction factor for thrust was added to a calculation spreadsheet and used to
produce the final results considering the elastic diagram data (the set of points that correlate the specimen deformation with the strength applied to it during the biomechanical test). This protocol was first described by Raghavan et al in 1996.23 A specimen may be tested only once in the case of destructive uniaxial tensile testing. Only one researcher (J.M.) performed all mechanical tests. Mechanical test calculations. The force extension data from the mechanical testing were used to determine the peak load (Ff) and extension at peak load (Lf L0). The failure properties calculated were failure strain, failure tension, and failure stress, as follows.26 Failure strain (extension at peak load as a fraction of original length): Df ¼ L f L 0 L 0 Failure tension (peak load per unit width of specimen strip): pffiffiffi Ff W f ¼ Ff W 0 1 þ Df Failure stress (peak load per unit cross-sectional area of specimen strip): Ef ¼ Ff A f ¼ Ff W 0 T0 1 þ Df
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Fig 1. A, Aneurysmal wall remaining postinterposition of synthetic prosthesis. B, Removal of the fragment with the largest possible dimensions, being the greatest dimension coincident with the longitudinal axis of the aorta.
Where L0, W0, and T0 are the length, the width, and the thickness of the specimen strip between the clamps at zero load. The Ef term in the above equations account for the reduction in cross-sectional area in case of stress calculations and reduction of width for tension calculations. Histologic analysis. The second specimen from the same patient, contiguous from the first, was used for histologic analysis in the Pathology Laboratory of Instituto do Coração Hospital. It was preserved in formaldehyde, paraffin-embedded, and cut in 5-mm slices. The slides were prepared, always by the same laboratory technician, for analysis of the inflammatory activity and count of collagen, elastic, and smooth muscle fiber, stained with hematoxylin and eosin, Masson trichrome, Picrosirius, Verhoeff-van Gieson, and for immunohistochemistry using smooth muscle actin antibody (M0851; Dako Cytomation, Glostrup, Denmark) and brown diaminobenzidine chromogen. The same biologist/laboratory technician read all immunohistochemistry slides. The slides, with different dyes corresponding to one specimen, were marked with a line in three identical adjoining sites, transversally, through superposition. Next, the reading of each slide was performed in the field obtained with optical magnification five times to the left of the previously marked locations. Hematoxylin and eosin staining allowed inflammation intensity evaluation; the semiquantitative analysis considered 0 points for inflammation absence, 1 point for mild (small foci with few and scattered cells), 2 points for moderate (less foci and number of cells), or 3 intense (big foci or numerous scattered cells). One biologist made all inflammatory activity analyses. The image obtained by microscopy was captured by a 3CCD video camera Donpisha (model XC 003; Sony, Tokyo, Japan), coupled to the microscope, and transmitted to the computer with the program Image Analysis System from Leica (Quantimet-500; Leica Microsystems, Wetzlar, Germany). The percentage of the elements under study on the slide with their staining was obtained by manual demarcation of the element of interest and quantified by the software. The result for each element corresponds to the arithmetic mean of the percentage measured in each of three adjacent fields (by the same researcher, J. M. for all cases). Statistical analysis. Categorical variables are presented in tables with absolute (n) and relative (%) frequencies. The
association between them was evaluated with the c2, Fisher exact, or with the likelihood ratio tests. Quantitative variables were presented descriptively in tables containing means, standard deviations, or medians, and first and third quartiles. The means of parametric variables were evaluated with the Student t-test or with analysis of variance. When significant, we used the Tukey test to discriminate differences. The distributions of nonparametric variables were assessed with the Mann-Whitney or Kruskal-Wallis analysis. When these were significant, we used the Dunn test to discriminate differences. P values of <.05 were considered statistically significant.32 The hypothesis checked was that the existence of differences in resistance and/or in composition among fragments from aneurysms larger or equal to or smaller than 5.5 cm and between symptomatic and asymptomatic aneurysms. RESULTS During the study period, 112 patients operated with AAA in our service were included. However, five AAA were inflammatory and were excluded during surgery. Specimens collected from the remaining 107, allowing prosthesis implantation and adequate coverage, were considered. During mechanical tests, 17 cases were further excluded because specimens slipped or ruptured close to the device clamps (up to 2 mm far from the clamps). Therefore, this study was completed with 90 cases. Among the 90 patients, 66 (73.3%) were men, 74 (82.2%) had hypertension, 38 (42.2%) had dyslipidemia, 7 (7.77%) had diabetes, and 78 (86.66%) were active or exsmokers. Aneurysms were 3.5-12 cm in diameter (mean: 6.54 6 1.66 cm), and 45 patients (50%) were asymptomatic. Among the 45 patients with symptoms, 10 aneurysms were ruptured. Aneurysms were divided into two groups, those with a MTD smaller than 5.5 cm and those with a MTD equal to or greater than 5.5 cm, and the association between the size category and demographic or clinical characteristics of the patients was searched. Only age was significantly associated with MTD: individuals with smaller aneurysms were significantly younger (P ¼ .049). Sex and prevalence of diabetes, hypertension, coronary artery disease, dyslipidemia, tobacco use, and family antecedents for AAA were not different.
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Table II. Physical and biomechanical measurements and histologic and histochemical properties (mean 6 standard deviation) according to the MTD of AAA Variable Thickness, mm Mean diameter, cm Failure load, N Failure strain Failure stress, N/cm2 Failure tension, N/cm Collagen area % (Masson) Collagen area % (Picrosirius) % Alpha actin/vessel % Alpha actin/adventitia Elastin area % (Verhoeff-van Gieson)c
MTD < 5.5 cm (n ¼ 25)
MTD $ 5.5 cm (n ¼ 65)
P
1.53 6 0.42 4.89 6 0.53 4.1 6 2.41 0.37 6 0.14 77.03 (53.32-111.71) 10.82 6 6.48 45.32 6 13.73 62 (56-70.5) 3.17 (1.08-6.22) 3.04 (2.49-4.22) <1
1.58 6 0.41 7.28 6 1.45 5.32 6 2.07 0.41 6 0.12 103.02 (75.70-144.42) 13.83 6 5.58 43.97 6 12.06 61 (56-67) 1.99 (1.48-4.22) 2.74 (1.47-4.2) <1
.662a .019a .260a .027b .020a .648a .350b .361b .322b
AAA, Abdominal aortic aneurysm; MTD, maximum transverse diameter. a 2 c test. b Mann-Whitney test. c Less than 1% were considered trace elements.
Table III. Physical and biomechanical measurements and histologic and histochemical properties (mean 6 standard deviation) according to the presence of symptoms in patients with AAA Variable
Asymptomatic (n ¼ 45)
Ruptured and symptomatic (n ¼ 45)
P
Mean diameter, cm Thickness, mm Failure load, N Failure strain Failure stress, N/cm2 Failure tension, N/cm Collagen area % (Masson) Collagen area % (Picrosirius) % Alpha actin/vessel % Alpha actin/adventitia Elastin area % (Verhoeff-van Gieson)c
6.09 (5.30-6.75) 1.57 6 0.41 4.65 6 2.05 0.41 6 0.13 84.76 (64.08-121.05) 12.81 6 5.77 44.02 6 13.79 62.00 (56.00-69.50) 2.35 (1.52-5.86) 2.81 (1.54-3.50) <1
7.15 (5.70-8.60) 1.56 6 0.41 5.32 6 2.36 0.38 6 0.12 103.02 (75.63-147.99) 14.08 6 6.11 44.67 6 11.17 60.00 (55.00-65.50) 2.52 (1.14-5.91) 3.10 (2.18-5.77) <1
.009a .848b .155b .287b .144b .154b .808b .120a .751a .169a
AAA, Abdominal aortic aneurysm a Mann-Whitney test. b Student t-test. c Less than 1% were considered trace elements.
The results from the biomechanical tests of AAA fragments were compared between larger and smaller aneurysms, and, as shown in Table I, the mean values of failure load, failure stress, and failure tension were significantly higher for larger AAA specimens. No differences were found comparing larger and smaller aneurysms regarding thickness or failure strain. Table II shows also the results from the histologic and histochemical analyses, but again no significant differences were found between larger and smaller aneurysms. The same demographic and clinical variables were investigated in relation to symptoms. The only clinical variable significantly associated with asymptomatic aneurysms was dyslipidemia (P < .001). Sex, age, and prevalence of other clinical variables were not different. The mean diameter of aneurysms was significantly larger among patients with symptoms or ruptured AAA, as shown in Table III. No other significant difference was found in histologic, histochemical, or biomechanical
variables comparing symptomatic or asymptomatic patients. In search for a discriminative variable, another analysis was made joining symptoms and AAA diameter, as described in Table IV. Considering specimens of smaller aneurysms from asymptomatic patients, specimen resistance was significantly lower, as shown by the failure load and failure tension variables. However, again, no histochemical feature could discriminate the lesions. Only inflammatory activity was shown to be higher in AAA with a larger diameter (category 3 of inflammatory activity MTD < 5.5 cm 28.6% MTD > 5.5 cm 70%; P ¼ .011) but with no differences regarding the presence of symptoms. DISCUSSION The present study analyzed fragments of the anterior wall of AAA removed during corrective open surgery and is the largest case series, according to our knowledge22-31 of destructive uniaxial biomechanical tests analyzed by
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1398 Tavares Monteiro et al
Table IV. Physical and biomechanical measurements and histologic and histochemical properties (mean 6 standard deviation) according to the presence of symptoms in patients with AAA and the MTD of aneurysms
Variable
Asymptomatic MTD < 5.5 cm (n ¼ 15)
Symptomatic MTD < 5.5 cm (n ¼ 10)
Asymptomatic MTD > 5.5 cm (n ¼ 30)
Symptomatic MTD > 5.5 cm (n ¼ 35)
P
Mean diameter, cm 5.21 6 0.56 4.84 6 0.50 6.67 6 0.86 7.68 6 1.51 Thickness, mm 1.39 6 0.3 1.75 6 0.49 1.66 6 0.44 1.5 6 0.37 Failure load, N 3.43 6 1.9 5.09 6 2.83 5.25 6 1.88 5.38 6 2.26 Failure strain 0.37 6 0.13 0.37 6 0.16 0.43 6 0.13 0.39 6 0.1 Failure stress, N/cm2 77.03 (34.82-102.77) 85.90 (57.05-129.13) 90.06 (70.38-130.99) 104.69 (82.58-151.89) Failure tension, N/cm 9.15 6 4.90 13.34 6 7.94 13.84 6 5.59 14.29 6 5.60 Collagen area % (Masson) 43.87 6 15.09 47.5 6 11.82 44.1 6 13.36 43.86 6 11.02 Collagen area % (Picrosirius) 62.00 (61.00-67.00) 56.00 (53.00-74.00) 61.00 (55.25-73.75) 60.00 (56.00-65.00) % Alpha actin/vessel 4.92 (1.32-6.22) 2.77 (1.00-7.50) 2.02 (1.50-3.17) 1.74 (1.27-5.91) % Alpha actin/adventitia 3.16 (2.23-3.55) 2.97 (2.44-7.56) 2.16 (1.46-3.06) 3.68 (1.64-5.55) Elastin area % (Verhoeff-van <1 <1 <1 <1 Gieson)e
.056a .029a,b .354a .097a,c .036a,d .871a .367a .702a .306a
AAA, Abdominal aortic aneurysm; MTD, maximum transverse diameter. a Kruskal-Wallis test. b Specimens from asymptomatic patients with AAA smaller than 5.5 cm had significantly lower values than asymptomatic patients with greater lesions and symptomatic patients with greater lesions. c Specimens from asymptomatic patients with AAA smaller than 5.5 cm had significantly lower values than symptomatic patients with greater lesions. d Specimens from asymptomatic patients with AAA smaller than 5.5 cm had significantly lower values than symptomatic patients with greater lesions. e Less than 1% were considered trace elements.
a clinical, morphologic, and histologic point of view; data obtained from 90 cases were considered appropriate for all evaluations. The use of methods consecrated by Raghavan23 in 1996 and the correlation of these results with those of a previous study conducted by the same group, give consistency to the findings (Table V).33,34 For a better comprehension of our line of reasoning, we chose four different cases in our sample, notwithstanding isolated patients, to reflect the behavior of the groups to which they belong (from larger and smaller AAA, symptomatic or asymptomatic) and also chose one tissue sample from a normal cadaveric abdominal aorta (without aneurysm) from another study in progress conducted by the same group in accordance with values reported for biomechanical properties of nonaneurysmal aorta in a previous study involving 26 individual cadavers (Table V).34 We used the same methodology to study normal cadaveric aortas and aneurysmal segments of surgical patients. The specimen relative to normal aorta was obtained from a cadaver because it would not be possible to obtain it from patients undergoing surgery, unlike the specimens regarding aneurysmal aorta. With these, we included Fig 5 with the respective elastics diagrams. As shown in Fig 5 and Table IV, as we moved from the group of small and asymptomatic aneurysms to the group of large and symptomatic aneurysms, we observed an increase in the maximum failure strength and failure tension of fragments associated with similar values of failure strain characterizing greater stiffness. As they increased in diameter, corresponding specimens were stiffer and more resistant; possibly as the aneurysm grows, the wall adapts, giving the tissue conditions to withstand larger loads. In this study, it seems that the MTD was more of a determinant of the biomechanical characteristics of the AAA than the patients’
Table V. Table comparing values found in this study for anatomic, biomechanical, and histologic parameters for fragments of the anterior wall of AAA and values obtained in other studies conducted by the same group for fragments of the anterior wall of corpses and AAA and ANA from autopsy
Age Diameter, cm Thickness, mm Failure strain Failure stress, N/cm2 Failure tension, N/cm Collagen % Masson Elastin % Verhoeff Smooth muscle fibers %
AAA surgery (n ¼ 90)
AAA autopsy (n ¼ 13)
69 6.62 1.57 0.39 103.1 13.1 44.37/61.91 <1 4.9
74 5.25 1.60 0.38 96.8 11.4 55.2 4.6 7.2
ANA autopsy (n ¼ 26) 62 1.42 0.47 137.2 14.7 49.3 17.1 30.2
AAA, Abdominal aortic aneurysm; ANA, normal aorta.
symptoms. Although behaviors were distinct from each other, specimen from AAA showed similar behavior compared with the specimen from the normal aorta: lower failure strain, lower resistance, and greater stiffness. These behaviors can be explained, at least in part, by our findings on histologic analysis. We observed also that the percent of elastic and smooth muscle fibers were both very reduced: less than 1% and around 2.5%, respectively, in small and large aneurysms. This could be explained by the fact that degradation of elastic and smooth muscle fibers happen very early in the course of aneurysmatic disease, with the steepest decline in the first stages.20 After the first aneurysmatic dilatation, structural or conformational change of the wall would influence the
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Tavares Monteiro et al 1399
Fig 5. Elastic diagram of individual specimens of abdominal aortic aneurysms (AAA) obtained in vivo and normal aorta from a cadaver: load (N) in the vertical axis and distension (mm) in the horizontal axis. The purple, green, yellow, and red lines are from specimens from larger or smaller individual AAA used in this study from asymptomatic (AST) or symptomatic (ST) patients. The blue line shows data from the same biomechanical test with tissue from normal, not aneurysmatic aorta from a cadaver.
acting forces of flow and hemodynamics with more radial and circumferential stress,35 leading to progressive expansion and loss of integrity36,37; remodeling would be an important physiological response to these changes.35 In aneurysms of different sizes, an enlargement of the arterial wall volume (ie, an increase in the diameter and surface with preservation of the thickness) and preservation of the percent of collagen fibers reveal an increase in the absolute mass of collagen, supporting the thought that the expansion of the AAA is followed by the wall remodeling. The concurrent increase in the arterial wall volume, maintenance of collagen fibers percent, and reduction in the elastic and smooth muscle fibers in AAA compared with normal tissue inspires the hypothesis that other cellular components (not studied here), such as the extracellular amorphous matrix, the quality of reposition material, and the final structure, should influence the function of the tissue. Fig 5 shows the behavior of the asymptomatic and small aneurysm (red line), which already has a reduced count of elastic and smooth muscle fibers and a reduced thickness of the wall, reflecting lower degree of remodeling and, consequently, less resistance to force and distension. This is consistent with the idea of elasticity loss as an early event after the first aneurysmatic dilatation. The small, symptomatic AAA (green line in the Fig 5) can be exposed to more stimuli, influencing remodeling and thickness growth earlier at the presence of symptoms. Cellular infiltrate count in AAA wall evidenced that larger aneurysms had an increased inflammatory activity in this study. However, even using a larger sample, this study
could not find a statistically significant association between inflammatory activity and the presence of symptoms, although there were a larger percentage of asymptomatic cases in category 3 of inflammation. Possibly, inflammation is not a marker for AAA rupture, but a stimulus to wall remodeling. Certain types of cells composing the AAA wall would be more or less responsive to mechanical stimuli, changing the composition of the wall tissue,38 and this could also happen regarding inflammatory activity, as a marker of reinforcement and not deterioration of the wall. The fact that larger aneurysms are more likely to rupture, as shown in other studies,1-3 does not mean that they have fragile walls compared with aneurysms with smaller diameter. The remodeling response has been shown to be heterogeneous even in studies by the same research team,26,30 with wide intervals for the biomechanical variables in different sites of the same aneurysms and in different aneurysms. Besides, the rupture event is one that arises from the interaction of two components: wall strength and the stress generated on it by external elements. An aneurysm of greater diameter, that theoretically shows a more resistant wall, could be exposed to more intense stress.11,39 Aneurysms with less resistant walls can be exposed to forces and stresses smaller and are, thus, less prone to breakage. One limitation of the present study is the use of fragments just from the anterior wall of AAA. Tissue remodeling is a heterogeneous process, with different regions of the same aneurysm showing different resistances40 in a manner that biomechanical data from one wall facet and
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extrapolated to all facets of the aorta are limited in offering tools for rupture risk calculation. However, in study with operated patients, the use of posterior, lateral, and anterior faces of the wall is not possible, once we must close the remaining aortic wall over the prosthesis to obtain proper treatment.15 However, the site for specimen collection was standardized in this study for all cases.9,27 Additionally, the identification of the site of rupture because of the hematoma and the need for rapid intervention did not allow dissection to locate this new site. This fact would require a longer intervention in a critical patient. The removal of the anterior fragment takes a short time. Another limitation to consider is the use of uniaxial mechanical test; in vivo, the aneurysmatic wall is submitted to multiaxial loads, which lead to different stress situations.9,41,42 It is necessary to consider the complexity of the AAA wall. We are still searching for mathematical models to predict the biomechanical behavior of AAA. We have to feed the models with stress and distension test data to determine which uniaxial tests are viable as a standardized tool. In line with this, our study is the largest essay on the biomechanical and histologic properties of tissue from aneurysms collected in vivo, and we believe it helps to understand more of the physiology of such a complex structure as that of the AAA. CONCLUSIONS The fragments of the anterior midsection from larger aneurysms were more resistant to rupture than those from smaller AAA, with no tissue properties that could explain this phenomenon in the histologic or histochemical analyses. Larger aneurysms, at least in this anterior wall, may be stronger than smaller aneuryms. It could point toward regional differences (heterogeneity, localized pathologies) as an important player in aneurysm rupture. Uniaxial strain tests are an important tool for the comprehension of a complex behavior such as that from an aneurysmal aortic wall. However, these tests still have limitations in providing information that would allow the calculation of the risk of rupture of AAA. AUTHOR CONTRIBUTIONS Conception and design: JM, ES, MR, PP-L, MH, JO Analysis and interpretation: JM, ES, MR, PP-L, MH Data collection: JM, ES, MR, PP-L Writing the article: JM, ES, MR, PP-L, MH Critical revision of the article: JM, ES, MR, PP-L, MH, JO Final approval of the article: JM, ES, MR, PP-L, MH, JO Statistical analysis: JM, ES, MR Obtained funding: JM, ES, JO Overall responsibility: JM REFERENCES 1. Heng MS, Fagan MJ, Collier JW, Desai G, McCollum PT, Chetter IC. Peak wall stress measurement in elective and acute abdominal aortic aneurysms. J Vasc Surg 2008;47:17-22; discussion: 22.
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2. Wilson K, Bradbury A, Whyman M, Hoskins P, Lee A, Fowkes G, et al. Relationship between abdominal aortic aneurysm wall compliance and clinical outcome: a preliminary analysis. Eur J Vasc Endovasc Surg 1998;15:472-7. 3. Wilson K, Whyman M, Hoskins P, Lee AJ, Bradbury AW, Fowkes FG, et al. The relationship between abdominal aortic aneurysm wall compliance, maximum diameter and growth rate. Cardiovasc Surg 1999;7:208-13. 4. Mortality results for randomised controlled trial of early elective surgery or ultrasonographic surveillance for small abdominal aortic aneurysms. The UK Small Aneurysm Trial Participants. Lancet 1998;352:1649-55. 5. Lederle FA, Wilson SE, Johnson GR, Reinke DB, Littooy FN, Acher CW, et al. Immediate repair compared with surveillance of small abdominal aortic aneurysms. N Engl J Med 2002;346: 1437-44. 6. Brewster DC, Cronenwett JL, Hallet JW Jr, Johnston KW, Krupski WC, Matsumura JS. Joint Council of the American Association for Vascular Surgery and Society for Vascular Surgery. Guidelines for the treatment of abdominal aortic aneurysms. Report of a subcommittee of the Joint Council of the American Association for Vascular Surgery and Society for Vascular Surgery. J Vasc Surg 2003;37: 1106-17. 7. Simão da Silva E, Rodrigues AJ, Magalhães Castro de Tolosa E, Rodrigues CJ, Villas Boas do Prado G, Nakamoto JC. Morphology and diameter of infrarenal aortic aneurysms: a prospective autopsy study. Cardiovasc Surg 2000;8:526-32. 8. Darling RC, Messina CR, Brewster DC, Ottinger LW. Autopsy study of unoperated abdominal aortic aneurysm: the case for early resection. Circulation 1977;56(3 Suppl):II161-4. 9. Vorp DA. Biomechanics of abdominal aortic aneurysm. J Biomech 2007;40:1887-902. 10. Truijers M, Pol JA, SchultzeKool LJ, van Sterkenburg SM, Fillinger MF, Blankensteijn JD. Wall stress analysis in small asymptomatic, symptomatic and ruptured abdominal aortic aneurysms. Eur J Vasc Endovasc Surg 2007;33:401-7. 11. Vorp DA, Vande Geest JP. Biomechanical determinants of abdominal aortic aneurysm rupture. Arterioscler Thromb Vasc Biol 2005;25: 1558-66. 12. Tilson MD, Newman K. Rationale for molecular approaches to the etiology of abdominal aortic aneurysm disease. J Vas Surg 1992;15: 924-5. 13. Rasmussen TE, Hallet JW Jr, Tazelaar HD, Miller VM, Schulte S, O’Fallon WM, et al. Human leukocyte antigen class II immune response genes, female gender, and cigarette smoking as risk and modulating factors in abdominal aortic aneurysms. J Vasc Surg 2002;35:988-93. 14. Muluk SC, Gertler JP, Brewster DC, Cambria RP, LaMuraglia GM, Moncure AC, et al. Presentation and patterns of aortic aneurysms in young patients. J Vasc Surg 1994;20:880-6; discussion: 887-8. 15. Vande Geest JP, Wang DH, Wisniewski SR, Makaroun MS, Vorp DA. Towards a noninvasive method for determination of patient-specific wall strength distribution in abdominal aortic aneurysms. Ann Biomed Eng 2006;34:1098-106. 16. Di Martino E, Mantero S, Inzoli F, Melissano G, Astore D, Chiesa R, et al. Biomechanics of abdominal aortic aneurysm in the presence of endoluminal thrombus: experimental characterization and structural static computational analysis. Eur J Vasc Endovasc Surg 1998;15: 290-9. 17. Carmo M, Colombo L, Bruno A, Corsi FR, Roncoroni L, Cuttin MS, et al. Alteration of elastin, collagen and their cross-links in abdominal aortic aneurysms. Eur J Vasc Endovasc Surg 2002;23:543-9. 18. Watton PN, Hill NA. Evolving mechanical properties of a model of abdominal aortic aneurysm. Biomech Model Mechanobiol 2009;8: 25-42. 19. Jacob MP, Badier-Commander C, Fontaine V, Benazzoug Y, Feldman L, Michel JB. Extracellular matrix remodeling in the vascular wall. Pathol Biol (Paris) 2001;49:326-32. 20. Sakalihasan N, Heyeres A, Nusgens BV, Limet R, Lapière CM. Modifications of the extracellular matrix of aneurysmal abdominal aortas as a function of their size. Eur J Vasc Surg 1993;7:633-7.
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21. Mohan D, Melvin JW. Failure properties of passive human aortic tissue. I‒uniaxial tension tests. J Biomech 1982;15:887-902. 22. He CM, Roach MR. The composition and mechanical properties of abdominal aortic aneurysm. J Vasc Surg 1994;20:6-13. 23. Raghavan ML, Webster MW, Vorp DA. Ex vivo biomechanical behavior of abdominal aortic aneurysm: assessment using a new mathematical model. Ann Biomed Eng 1996;24:573-82. 24. Thubrikar MJ, Labrosse M, Robicsek F, Al-Soudi J, Fowler B. Mechanical properties of abdominal aortic aneurysm wall. J Med Eng Technol 2001;25:133-42. 25. Vorp DA, Lee PC, Wang DH, Makaroun MS, Nemoto EM, Ogawa S. Association of intraluminal thrombus in abdominal aortic aneurysm with local hypoxia and wall weakening. J Vasc Surg 2001;34:291-2. 26. Raghavan ML, Kratzberg J, Castro de Tolosa EM, Hanaoka MM, Walker R, da Silva ES. Regional distribution of wall thickness and failure properties of human abdominal aortic aneurysm. J Biomech 2006;39:3010-6. 27. Di Martino ES, Bohra A, Vande Geest JP, Gupta N, Makaroun MS, Vorp DA. Biomechanical properties of ruptured versus electively repaired abdominal aortic aneurysm wall tissue. J Vasc Surg 2006;43: 570-6; discussion: 576. 28. Vande Geest JP, Dillavou ED, Di Martino ES, Oberdier M, Bohra A, Makaroun MS, et al. Gender-related differences in the tensile strength of abdominal aortic aneurysm. Ann N Y Acad Sci 2006;1085:400-2. 29. Xiong J, Wang SM, Zhou W, Wu JG. Measurement and analysis of ultimate mechanical properties, stress-strain curve fit, and elastic modulus formula of human abdominal aortic aneurysm and nonaneurysmal abdominal aorta. J Vasc Surg 2008;48:189-95. 30. Raghavan ML, Hanaoka MM, Kratzberg JA, de Lourdes Higuchi M, da Silva ES. Biomechanical failure properties and microstructural content of ruptured and unruptured abdominal aortic aneurysm. J Biomech 2011;44:2501-7. 31. Reeps C, Maier A, Pelisek J, Härtl F, Grabher-Meier V, Wall WA, et al. Measuring and modeling patient-specific distributions of material properties in abdominal aortic aneurysm wall. Biomech Model Mechanobiol 2013;12:717-33. 32. Rosner B. Fundamentals of biostatistics. 4th ed. New York: Duxbury Press; 1994.
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33. Raghavan ML, Ikeda MH, Silva ES. Failure strength of abdominal aortic aneurysm: a necropsy study. New Orleans, LA: Proceedings of the ASME International Mechanical Engineering Congress and Exposition. Advances in Bioengineering; November 17-22 2002. 34. da Silva ES, et al. Oral Presentation. Sotckholm, Sweden: Proceedings of the Endovascular Surgery-Bringing Basic Science into Clinical Practice. Session 1-Basic Mechanism: Biomechanical aspects of abdominal and thoracic aorta according to the age; March 19-21, 2009. 35. Astrand H, Rydén-Ahlgren A, Sandgren T, Länne T. Age-related increase in wall stress of the human abdominal aorta: an vivo study. J Vasc Surg 2005;42:926-31. 36. Speelman L, Bosboom EM, Schurink GW, Hellenthal FA, Buth J, Breeuwer M, et al. Patient-specific AAA wall stress analysis: 99-percentile versus peak stress. Eur J Vasc Endovasc Surg 2008;36: 668-76. 37. Salsac AV, Sparks SR, Lasheras JC. Hemodynamic changes occurring during the progressive enlargement of abdominal aortic aneurysms. Ann Vasc Surg 2004;18:14-21. 38. Humphrey JD, Na S. Elastodynamics and arterial wall stress. Ann Biomed Eng 2002;30:509-23. 39. Di Martino ES, Guadagni G, Fumero A, Ballerini G, Spirito R, Biglioli P, et al. Fluid-structure interaction within realistic threedimensional models of the aneurysmatic aorta as a guidance to assess the risk of rupture of the aneurysm. Med Eng Phys 2001;23:647-55. 40. De Martino RR, Nolan BW, Goodney PP, Chang CK, Schanzer A, Cambria R, et al. Outcomes of symptomatic abdominal aortic aneurysm repair. J Vasc Surg 2010;52:5-12.e1. 41. Volokh KY. Comparison of biomechanical failure criteria for abdominal aortic aneurysm. J Biomech 2010;43:2032-4. 42. Vande Geest JP, Sacks MS, Vorp DA. The effects of aneurysm on the biaxial mechanical behavior of human abdominal aorta. J Biomech 2006;39:1324-34.
Submitted Dec 11, 2012; accepted Apr 29, 2013.
Additional material for this article may be found online at www.jvascsurg.org.
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Fig 2 (online only). A, Traction device Instron 2200 associated with Palm Top with Inspec software for control movements of the traction device. B, Laptop with Series IX software for data management.
Fig 3 (online only). Performing the test over immersion to preserve the moisture conditions.
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Fig 4 (online only). As occurred the motion of head traction, it emerges from the tub, thus reducing the volume of liquid displaced by it and consequently reducing the thrust that acted on it.