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Biology Articles » Cryobiology » An in vitro study of cryopreserved and fresh human arteries: a comparison with ePTFE prostheses and human arteries studied non-invasively in vivo » Materials and methods

Materials and methods
- An in vitro study of cryopreserved and fresh human arteries: a comparison with ePTFE prostheses and human arteries studied non-invasively in vivo

Non-invasive human studies

Normotensive subjects (6 males, 4 females), 50 ± 13 years old, body mass index 25 ± 1 kg/m2 (Group I, n = 10) were used in this study. All subjects gave written consent for this non-invasive clinical research. Diameter and wall thickness measurements were performed using echographic techniques with real-time B-mode ultrasound imaging (Hewlett-Packard Sonos 1500). The right common carotid artery, 3 cm proximal to the bifurcation of the vessel, was examined with a 7.5 MHz probe. Scanning of the common carotid artery was performed in the antero-posterior projection, with the subject lying on his back [3], [4] and [13]. During scanning, the sound beam was adjusted perpendicular to the arterial surface of the far wall of the vessel to obtain two parallel echogenic lines corresponding to the lumen-intima and media-adventitia interfaces. Once the two parallel echogenic lines of the far wall were clearly visible on the monitor, along at least 1 cm of the segment to be measured, two kinds of image acquisition were performed. These were [1] a fixed image with end-diastolic electrocardiogram triggering, obtained from surface electrodes to assess intima-media thickness; and [2] a sequence of images to determine the instantaneous waveform of the arterial diameter. The images were digitized into 640 × 580 pixels with 256 gray levels and stored in a mass memory system. Three acquisitions were performed at the same anatomical site and the electrocardiogram signal was simultaneously acquired. These signals were analyzed off-line with appropriate software (Iôtec System, Paris, France). This software was based on the analysis of gray level density and on specific tissue recognition algorithms [4] and [13]. To obtain the diameter waveform, the sequence of images was analyzed automatically frame by frame. The anterior and posterior walls were detected. After analyzing the overall sequence, the software yielded as output the internal diameter waveform, calculated as the difference between the movement of the far and near wall. This methodology has been previously validated against sonomicrometry [14]. Arterial pressure waveform measurements were recorded at the same site as the diameter wave after the echographic recording. A probe that incorporated a Millar micromanometer in its tip, and had the same high-frequency response as the conventional Millar catheter, was used to measure arterial pressure waveform. The instantaneous pressure waveforms of four cardiac cycles were digitized every 1 ms and the signal average calculated. The common carotid artery pressure signal, obtained with the probe, was calibrated by assigning to its minimum value, the minimum value measured by brachial sphygmomanometry and to its mean value, the mean brachial pressure, calculated as 1/3 of pulse pressure plus diastolic pressure. We assumed that mean pressure did not change in large conduit arteries and that diastolic pressure (as opposed to systolic pressure) did not substantially differ between the brachial and the carotid artery [3].

During both arterial diameter and pressure measurements, the spikes corresponding to the QRS complex of the electrocardiogram were acquired and stored together with the diameter and pressure signals. The pressure and diameter waveforms were identified according to the QRS complex of the electrocardiogram. Each signal was interpolated in time to obtain the same number of data points, allowing calculation of the averaged cardiac cycle.

In vitro studies

Tissue procurements were performed according to the Guide of the Transplant Program of the National Organ and Tissue Bank of Uruguay. All procedures of vascular tissue procurement and processing conformed with the ethical and safety requirements for therapeutic use, including written consent. The general exclusion criteria were those specified by the International Atomic Energy Agency (IAEA: International Standards for Tissue Banks). the donor age was 23–45 years (mean = 29.6 years). Right and left common carotid arteries taken from seven donors following certification of brain death, and were procured by a surgical, aseptic technique during the harvesting of multiple organs and tissues. After aortic clamping and cardiac arrest, aseptic cleaning of the carotid region was carried out with 7% Povidone-iodine solution. Sterile drapes were used to establish the surgical field. Skin incision was made along the anterior border of the sternocleidomastoideus muscle. Each common carotid artery was carefully cleared from the surrounding tissues after moving the sternocleidomastoideus muscle laterally with forceps. In situ measurements of the length of the common carotid were performed (5 cm) and two sutures were placed in the adventitial tissue of the vessel for reference. Afterwards all segments were cut and removed. After harvesting in each donor, both common carotid arteries (right and left) were washed with saline solution and stored at 4 °C. The warm ischemia time was 55–66 min (mean = 61 min). Cold ischemia was maintained in all cases for 4 days. Arteries to be used as fresh-controls (Group II, n = 7) were sent to the biomechanical laboratory and arteries to be cryopreserved (Group III, n = 7) were immersed in the cryopreservation solution. This solution consisted of RPMI 1640 (85%), human albumin solution (5%) and dimethyl sulphoxide (Me2SO, 10%), and was contained in a thermally sealed cryo-resistant bag, in a laminar flow cabinet [10]. Our cryopreservation program was carried out using in a controlled-rate freezing system (Model 9000—Gordinier Electronics, 29975 Parkway—Roseville, MI 48066, USA). First, a low programmed cooling rate was used (mean value of 1 °C/min) to −90 °C [24]. Then, rapid cooling from −90 to−142 °C was performed by the immediate transfer of the bag containing the tissue into the gaseous phase of the liquid nitrogen storage refrigerator [24]. Frozen arterial specimens were stored for 30 days at −142 °C in the vapour above the liquid nitrogen level (Mark III, Temperature and Liquid Level Controller, Taylor, Wharton. Theodore, Alabama, USA). On the last day of the storage period (the 30th day) the vessels were warmed to 20 °C during 30 min and then immersed in warm water at 40 °C until totally defrosted [10]. Once thawed, the Me2SO was removed by rinsing in four serial dilutions at 20 °C, each step reducing the concentration by 50%, with 10 min at each step. The arteries to be used in the cryopreserved group were sent to the biomechanical laboratory [8].

Each common carotid artery (fresh or cryopreserved) was non-traumatically mounted on specially designed cannulas that were connected to the in vitro flow circuit and immersed in oxygenated Tyrode’s solution at 37 °C and pH 7.4. The same fluid was perfused using a circuit consisting of polyethylene tubing and a Windkessel chamber that was powered by a pneumatic pump (Fig. 1). The pneumatic device was regulated by an air supply that allowed fine adjustments of pump rate, pressure values and waveforms [8]. Each 5 cm segment was instrumented with a pressure microtransducer (1200 Hz frequency response, Konigsberg Instruments, Pasadena, CA, USA) that was inserted into the vessel through a stab wound. Each pressure transducer had previously been calibrated against a mercury manometer. To measure the external arterial diameter a pair of ultrasonic crystals (5 MHz, 2 mm diameter) was sutured to the adventitia of the common carotid artery. The transit time of the ultrasonic signal (1580 m/s) was converted into distance by means of a sonomicrometer (1000 Hz frequency response, Triton Technology, San Diego, CA, USA). Optimal positioning of the dimensional gauges was assessed by oscilloscope. Finally, ePTFE graft segments (thin wall, 6 mm ID, Gore-Tex Vascular graft, W.L. Gore and Associates, Flagstaff, Arizona, USA) were mounted on the in vitro system (Group IV, n = 6). To characterize the mechanical behavior of the ePTFE grafts they were also instrumented with a pressure microtransducer and a pair of ultrasonic crystals, using a similar procedure to that used for the common carotid artery. Once the arterial or prosthetic grafts were placed in the specimen chamber, the segments were allowed to equilibrate for a period of 15 min under steady flow conditions (150 ml/min). The mean pressure was between 80 and 85 mmHg and the stretching rate was 80 beats/min (1.34 Hz). (Fig. 2

Data acquisition

After a period of mechanical stability, a similar measurement protocol was applied for the fresh carotid common artery segments, the cryopreserved arteries and the ePTFE conduits. Diameter and pressure waves were measured under dynamic conditions, displayed in real-time, digitized every 5 ms and stored on the computer hard disk for off-line analysis. The segments were submitted to a steady state flow of 150 ml/min, a stretching rate of not, vert, similar80 cycles/min, and an intravascular pulse pressure of not, vert, similar40–45 mmHg with a mean pressure of 80–85 mmHg. Flow, pressure, and pump rate levels were chosen to be similar to the observed values in normotensive subjects. Between 10 and 20 consecutive beats were sampled and analyzed.

After the measurements had been made, the segment was weighted to estimate the wall thickness. A computerized procedure was used to determine the stress-strain loop and to calculate the mechanical parameters using special software developed in our laboratory [2].

Data analysis

As we have described above, signals were assessed by two different approaches: (a) non-invasive in vivo measurement of pressure and diameter were obtained from the right common carotid arteries in human subjects, and (b) in vitro measurements of pressure and diameter signals were obtained from ePTFE segments, fresh and cryopreserved common carotid artery segments that had been obtained from multiorgan donors. The in vitro wall thickness of common carotid artery segments and ePTFE segments was calculated as the difference between the external radius (Re = external diameter/2), and the internal radius (Ri), estimated according to the previously reported technique [2]. The wall thickness of the normotensive subjects’ arteries was assumed to be similar to the intima media thickness index [5]. Strain (ε) and circumferential wall stress (σ) were calculated according to previous work [2]. The viscoelastic properties of arterial segments were studied assuming the Kelvin–Voigt viscoelastic model (spring-dashpot). According to this, σ developed in the wall (σtotal) can be divided into an elastic component (σelastic) and a viscous component (σviscous) [2] thus:-

σtotal=σelastic+σviscous (1)

The viscous component is proportional to the first derivative of ε with respect to time (dε/dt). Consequently, σelastic can be expressed as:

σelastic=σtotal-η.dε/dt, (2)

where η is the viscous modulus of the arterial wall. To obtain the pure elastic σ component, the viscous term must be subtracted from the σtotal. This was performed by minimizing the area of the σ-ε hysteresis loop while still preserving the clockwise direction of the loop [2]. Once the elastic component of σTotal had been isolated, the elastic modulus (E) was calculated from the slope of the linear regression curve, evaluated at the mean prevailing pressure. Theoretically the elastic modulus describes the inherent stiffness of the vessel, independent of its geometry. The slope of the relationship between stress and the first derivative of strain (strain velocity) is the viscous modulus which is strongly related to the viscous losses in the wall.

A variety of elastic parameters, commonly used in clinical work have also been computed.

The pressure-strain or Peterson modulus (EP), which describes the fractional pulsatile diameter change that occurs in an artery exposed to a given change in intra-luminal pressure, was calculated as [22]:


 where Ds and Dd are the systolic and diastolic diameter values, respectively, and Ps and Pd are the corresponding systolic and diastolic pressure values.

The stiffness index (SI) may provide a more reliable measurement of the arterial wall stiffness, particularly at low physiological pressures. It was calculated as [22]:


The pulse wave velocity (PWV) is the wave speed which can be related to the conduit wall elasticity via the Moens–Korteweg equation:


where E is the elastic modulus, ρT is the blood density (ρT = 1.06 g/cm3), hm is the mean wall thickness, and Rm is the mean radius.

Statistical analysis

All values were expressed as means ± standard deviation (means ± SD). Comparisons were made using ANOVA followed by a Bonferroni test. To indicate significant differences among groups, the criterion P 

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