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Cell Biology International (2006) 30, 439–444 (Printed in Great Britain)
Design of compliance chamber and after-load in apparatus for cultured endothelial cells subjected to stresses
Hao Dingab, Aike Qiaoa, Lixing Shenb, Mingyang Lib, Zhenglong Chenb, Xiaojun Yuc and Yanjun Zengac*
aBiomechanics & Medical Information Institute, Beijing University of Technology, No. 100 PingLeYuan, Beijing 100022, China
bThe Medical Instrumentation College, University of Shanghai for Science and Technology, Shanghai 200093, China cMedical College of Shantou University, Shantou 515031, China Abstract In order to create a hemodynamic environment that can simulate the physiological condition of arteries, an in vitro experiment apparatus was designed whose key modules were compliance chamber and after-load. These two modules were developed based on the theories of hemodynamics. Both the normal and shear stress to which endothelial cells are exposed can be controlled with these modules, thus facilitating the research of endothelial cells subjected to stresses. Keywords: Biomechanics, Hemodynamic environment, Experiment, Simulation, Blood flow, Artery. *Corresponding author. Biomechanics & Medical Information Institute, Beijing University of Technology, No. 100 PingLeYuan, Beijing 100022, China. Tel.: +86 10 67391685. 1 Introduction Endothelial cells (ECs), which form a layer of membrane covering heart valves and blood vessels, play a very important role in the physiological and pathological activities of the cardiovascular system. Changes in their structure and function are key events in the occurrence and development of some vascular diseases, such as hypertension and atherosclerosis (Dewey et al., 1981; Franke et al., 1984; Eskin et al., 1984; Helmlinger et al., 1991). ECs in vivo are always exposed to a hemodynamic environment. Many previous research studies have considered that shear stress of blood flow is the main factor influencing ECs (Frame et al., 1998; Passerini et al., 2003; Chen et al., 2003; Greisler et al., 1990; Brooks et al., 2002; Mu and Du, 2004). In fact, besides shear stress, normal stress (i.e. blood pressure) also acts on ECs. Under normal physiological conditions, normal stress, with magnitudes of 15,996/10,664 In view of the fact that it is impossible to measure the shear stress of blood flow in arteries both in vivo and in vitro, many in vitro experiments have been conducted to calculate shear stress indirectly (Liu et al., 2001). In order to create a realistic hemodynamic environment (including both shear stress and normal stress) to which the ECs are exposed, we developed an apparatus that can simulate steady and pulsatile blood flow phenomena in arteries. In this apparatus, the compliance chamber and after-load modules are two key modules for the simulation of in vitro experiments. The purpose of this paper is to show the design of these two important modules in the apparatus. 2 Methodology Technically, the compliance chamber and after-load should satisfy the following requirements: first, they are able to simulate normal human aorta systolic and diastolic pressure (systolic pressure: 15,996–23,994 2.1 Principle of the compliance chamber The function of the compliance chamber is to simulate elastic function of the aorta, and change the intermittent blood ejection of the pulsatile pump into a continuous pulsatile flow. The compliance chamber, which is composed of a gas chamber and a liquid chamber to simulate the elastic artery, stores and discharges energy for the test system. Its gas chamber volume can be calculated with (Zhang and Huang, 1991): (1) The total gas volume 2.2 Principle of the after-load The function of the after-load is to simulate the peripheral resistance of the micro-vessels and capillary vessels (“lump resistance model”). Blood pressure is the only normal stress acting on the ECs. The blood pressure level can be changed by adjusting the opening of the after-load. We have found that the throttle, as an effective load, plays a key role in adjusting peripheral resistance of the vascular system. The throttle is a “lump resistance model” whose role is not different from that of capillary vessels when studying large arteries in hemodynamics. Physiologically, the total length of capillary vessels is very long and the resistance distributes along the total length without converging at one point. The distribution of resistance is different depending on the difference in diameter, length and branch of capillary vessels. In this case, the “lump resistance model” has no effect when studying the behavior of capillary vessels. However, when studying the blood flow in large arteries far upstream from capillary vessels, it is not only feasible but also technically convenient to consider the influence of capillary vessels as “lump resistance”. As a preliminary study, we assume that the blood flow is a laminar flow in a rigid circular pipe. The shear stress acting on the ECs is just the shear wall stress of arteries. According to Poiseuille's law, parabolic velocity profile leads to wall shear stress (2) In our design, the test apparatus has a rectangular flow chamber whose height is much less than its width and where the ECs are planted. For Poiseuille flow in a rectangular chamber, the wall shear stress can be denoted as (3) Obviously, the shear stress is proportional to the flow rate. The flow rate in the pipe can be obtained from the following equation (Diao, 1991; Liu and Li, 1997): (4) The flow rate of the heart pump keeps constant if the output per pulse and the heart rate do not vary. Thus, the pressure drop is proportional to the resistance. Increasing R can induce the increase of 3 Implementation This apparatus consists of a pulsatile pump, a compliance chamber, a rectangular chamber, a reservoir, a thermostatic device, an after-load system and joint pipelines. A diagram of the testing circulation system is shown in Fig. 1. 
Fig. 1 Semantic of the testing circulation system. 3.1 Compliance chamber The compliance chamber (54 
Fig. 2 The structure (a) and photograph (b) of compliance chamber. Adjusting the ratio of gas to liquid (gas/liquid) changes the amplitude of pulsatile flow. Compressing the gas loading valve and boosting air into the gas chamber, the gas/liquid increases and the amplitude of pulsatile flow decreases. Turning on the purge valve, the gas/liquid reduces and the amplitude of pulsatile flow augments. 3.2 After-load The after-load (43 
Fig. 3 The structure (a) and photograph (b) of the after-load. 3.3 Specifications of the design The rectangular chamber in our test apparatus has an orifice of ∅24 (1) Systolic pressure is 15,996–23,994 Fig. 4 The structure of rectangular chamber. (2) Diastolic pressure is 7998–15,996 (3) The pump's flow rate must be in the range of 20–600 As mentioned above, the shear stress is proportional to the flow rate and varies within a certain range in the human body. If we want to obtain physiological shear stresses of 2–300 (4) Pulsatile frequency should be 40–200 Pulsatile frequency reflects heart rate, and the normal heart rate of man is 75 beats per min. But in order to simulate the normal heart rate and the abnormal one we have to choose a larger measuring range in this apparatus, therefore the value of pulsatile frequency is 40–200 (5) In order to make the pulsatile amplitude adjustable within a stated range, the total compliance chamber volume The cavity of the compliance chamber is 250 Table 1. Relationship between the value of gas/liquid and P2/P1
Analyzing the data in Table 1, we can obtain the following conclusion: first, when the value of gas/liquid changed, P 4 Results The working procedure of compliance chamber and after-load in the apparatus for cultured endothelial cells subjected to stresses designed in this paper is shown as follows. Not blood but nourishing fluids were employed in the apparatus to feed the ECs on the rectangular chamber. The liquid pumped by pulsatile pump is divided into two branches after the compliance chamber: one is connected to a throttle and then flows back to the reservoir, the other enters the rectangular chamber, and then flows back to the reservoir through the after-load (Fig. 1). This device can simulate the normal systolic and diastolic pressure of 15,996/10,664 
Fig. 5 Simulating the normal systolic pressure and diastolic pressure (15,996/10,664 
Fig. 6 Regulating the amplitude of pulsatile flow. 
Fig. 7 Regulating the absolute value of blood pressure. 5 Discussion Although the apparatus is able to accomplish our intention, it still has some drawbacks as follows: (1) The purpose of this apparatus is to create a hemodynamic environment that is similar to the physiological condition of arteries. In order to observe ECs' changing states more conveniently, three-dimensional flow field in the arteries is simplified into one-dimensional flow field in the rectangular chamber (Fig. 4), but the simplification creates a difference between the realistic condition and the experimental one. (2) The ECs are planted on an inflexible cover-slip, which is different to the elastic foundation base of arteries. (3) The shear stress is obtained indirectly from the calculation of pressure difference, and there is no direct and simple measurement at the present time. (4) The after-load mentioned by this paper is a “lump resistance model”, which simulates the resistance of capillary vessels to blood flow. However, the “lump resistance model” has no effect when studying the behavior of capillary vessels. 6 Conclusion Since the shear stress acting on vascular endothelium is inaccessible in vivo, we designed an in vitro experiment apparatus in which the compliance chamber and the after-load are two key modules in order to simulate the hemodynamic environment for cultured ECs subjected to both normal stress and shear stress. By adjusting these two modules, we can change the normal stress and the shear stress to which ECs are exposed. This apparatus is supplied as a trial production to Shanghai Cerebrovascular Disease Prevention Research Institute and Life Sciences College of Tongji University. Their experimental findings were inspiring and confirmed the practicability of the designed apparatus. Therefore, with this apparatus, more realistic hemodynamic conditions can be obtained for in vitro experiments using ECs under physiological conditions, thus facilitating the research of ECs subjected to stresses. Acknowledgments For significant contribution to this work, the authors wish to thank Prof. Shixiong Xu from Fudan University for his support and assistance. References Brooks AR, Lelkes, PI, Rubanyi, GM. Gene expression profiling of human aortic endothelial cells exposed to disturbed flow and steady laminar flow. Physiol Genomic 2002:9:1:27-41 Chen H, Wu, L, Liu, X, Chen, Y, Wang, B. Effects of laminar shear stress on IL-8 mRNA expression in endothelial cells. Biorheology 2003:40:1–3:53-8 Dewey CF, Bussolari, SR, Gimbrone, MA. The dynamic response of vascular endothelial cells to fluid shear stress. J Biomech Eng 1981:103:177-81 Diao Y. Principles and application of biomechanics. 1991: Eskin SG, Ives, CL, Mcintire, LV. Response of cultured endothelial cells to steady flow. Microvasc Res 1984:28:87-94 Franke RP, Grafe, M, Schnittler, H. Induction of human vascular endothelial stress fibers by shear stress. Nature 1984:307:648-9 Greisler HP, Johnson, S, Joyce, K, Henderson, S, Patel, NM, Alkhamis, T. The effects of shear stress on endothelial cell retention and function on expanded polytetrafluoroethylene. Arch Surg 1990:125:11:1622-5 Helmlinger G, Geiger, RV, Schreck, S. Effects of pulsatile flow on cultured vascular endothelial cell morphology. J Biomech Eng 1991:113:123-31 Liu Q, Cai, S, Wu, Y. A new calculating method for the dynamic wall shear stress of experimental vessel segments. Acta Biophys Sin 1997:13:3:507-11 Liu Z, He, F, Xu, G, Chen, Y. The determination of artery wall's shear stress. Sci China Ser A 2001:31:7:651-60 Liu Z, Li, X. The principle and method of hemodynamics. 1997: Frame MDS, Chapman, Gary B, Markino, Yoshia, Sarelius, Ingrid H. Shear stress gradient over endothelial cells in a curved microchannel system. Biorheology 1998:35:4:245-61 Mu Y, Du, G. Advance in the culture methods of endothelial cell in vitro. Foreign Med Sci Sect Pharm 2004:31:6:361-5 Passerini AG, Milsted, A, Rittgers, SE. Shear stress magnitude and directionality modulate growth factor gene expression in preconditioned vascular endothelial cells. J Vasc Surg 2003:37:1:182-90 Zhang H, Huang, Y. Hydraulic transmission. 1991: Received 28 November 2005/10 January 2006; accepted 14 February 2006 doi:10.1016/j.cellbi.2006.02.003 |
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355 Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB) |
Abstract
should be between 200 and 300
.
, we can obtain the following data (