Abstract:
The present invention in one aspect provides a bioreactor for vascular construct that comprises a pulse-generating device with simple structure and reliable and stable pulse-generating operation. In another aspect, the present invention provides bioreactor for vascular construct that implements rotation of vascular construct and culture chamber, axial stretching of vascular construct, and duplicating pulsatile flow.

Description:
FIELD OF THE INVENTION 
       [0001]    The invention is generally in the field of tissue engineering, more specifically in the field of tissue engineering bioreactor for vascular constructs with rotary, stretching, and perfusion functions. 
       BACKGROUND OF THE INVENTION 
       [0002]    Coronary and peripheral artery bypass grafting is commonly used to relieve the symptoms of angina and other vascular deficiencies. To date, autograft, allograft blood vessels, vascular xenograft, and synthetic materials can not be an ideal substitute for small diameter (&lt;6 mm) vascular grafts. Developing small diameter vascular grafts with high patency and durability as substitutes for the coronary and peripheral vasculature is a challenge for vascular tissue engineering. 
         [0003]    In recent years, construct tissue-engineered vascular with bioreactor may bring prospect to this area. Firstly bioreactors can be custom designed to engineer tissues with complicated three-dimensional geometry containing multiple cell types. Secondly bioreactor can supply a controllable biochemical and mechanical environment to promote cell growth, maturation, and tissue differentiation. At last, bioreactors can serve as tissue growth systems as well as packaging and shipping units that can be delivered directly to surgeons. The research of tissue engineering bioreactor for vascular construct focus on the following aspects: 
         [0004]    1. High-density cell seeding and uniform cell distribution on 3D scaffolds. High seeding density can enhance tissue formation, and uniform distribution of cells within the scaffold can significantly affect the tissue properties. Perfusion seeding has been reported to be a more effective way to improve both seeding efficiency and cell distribution than static seeding or the stirring-flasks bioreactor ( 2 - 20 ). Perfusion seeding bioreactors have been designed for engineering vascular grafts, cartilage, hepatocyte and cardiac tissues. 
         [0005]    2. Increase of mass transport. The rotating wall bioreactor can generate dynamic flow to improve nutrients and wastes transfer and to provide a low stress. Research results have shown that properties of engineered tissue cultured in the rotating wall bioreactor were superior to those of static or stirring-flask bioreactor ( 2 - 30 ). As the effect of the rotating wall bioreactor depended on the perfusion rate, the sheer stress, the balance of nutrients and wastes transfer, design and optimize the rotating wall bioreactor match the needs of specific tissues is important 
         [0006]    3. Mechanical stimulation. Many studies have shown that flow sheer stress had significant effect on endothelial cells; cyclical mechanical stretch was found to increase tissue organization and expression of elastin by smooth muscle cells seeded in polymeric scaffolds ( 2 - 52 ); pulsatile radial stress improved the mechanical strength of engineered blood vessels ( 2 - 53 ). 
         [0007]    Yuji Narita et al. designed a non-rotary wall and perfusion bioreactor for vascular construct (Novel Pulse Duplicating Bioreactor System for Tissue-Engineered Vascular Construct. Tissue Engineering 2004; 10(7-8):1224-1233.), in which a balloon was immersed in liquid confined in a solid chamber. Inflation of the balloon was modulated by an air-pump device to cause pulse-like pressure variation in the liquid confined in the chamber and in liquid in pipeline connected to the chamber. 
         [0008]    Craig A. Thompson et al. developed a perfusion bioreactor for vascular construct by using a mechanical ventilator to induce pulsatile, laminar flow into a fluid column. They claimed that their design can generate pressurized waveforms similar to mammalian physiology (A Novel Pulsatile, Laminar Flow Bioreactor for the Development of Tissue-Engineered Vascular Structures. Tissue Engineering 2002; 8(6): 1083-1088.). 
         [0009]    Boris A. Nasseri et al. designed a rotating bioreactor for vascular construct to improve mass transfer. A hybridization oven was used for rotational seeding and culture. Culture vessel was placed in the hybrization oven and was rotated around the central axis (Dynamic Rotational Seeding and Cell Culture System for Vascular Tube Formation. Tissue Engineering 2003; 9(2): 291-299.). 
         [0010]    Ralf Sodian et al. designed a non-rotating wall perfusion bioreactor for vascular construct (Tissue-Engineering Bioreactors: A New Combined Cell-Seeding and Perfusion System for Vascular Tissue Engineering. Tissue Engineering 2002; 8(5):863-870.), in which a pneumatic device was used to generate pulsatile flow. 
         [0011]    Chrysanthi Williams et al. described a non-rotating wall bioreactor to culture small diameter arterial constructs. With two peristaltic pumps the bioreactor provided dual perfusion flow through the lumen and on the external surface of the constructs (Perfusion Bioreactor for Small Diameter Tissue-Engineered Arteries. Tissue Engineering 2004; 10 (5-6):930-941.). The internal perfusion provided sheer stress and pulsatile flow environment. The external perfusion improved mass transfer. 
         [0012]    Satish, C. Muluk et al. designed a non-rotating wall bioreactor for vascular construct that implemented stretching of vascular tissue by a stretching motor and twisting of vascular tissue by a twisting motor (Enhancement of tissue factor expression by vein segments exposed to coronary arterial hemodynamics. Journal of vascular surgery: official publication, the Society for Vascular Surgery [and] International Society for Cardiovascular Surgery, North American Chapter 1998; 27(3):521-527). Vascular internal perfusion was implemented in this design. 
         [0013]    In summary, the existing tissue engineering bioreactors for vascular construct have some limitations: First, they cannot use simple mechanical stimuli with little consideration on the blood flow impedance, vascular compliance, and vascular inertia resistance to reproduce a similar flow environment in vivo; second, periodical axial tensile, cyclical stretch, twisting, and sheer stress can not be imposed on vascular construct at the same time; third, the properties of seeding efficiency, uniform cell distribution, and mass transfer need to be improved. For these reasons, it is essential to develop bioreactor for vascular constructs, which can provide physiological pulsatile flow perfusion and multi-mechanical stimulation. 
       SUMMARY OF THE INVENTION 
       [0014]    The present invention is to provide a multi-module tissue engineering bioreactor for vascular construct of different length and diameter. Also it provides devices and methods for research on the cellular, histological and mechanical properties of vascular constructs. The present invention is characterized by the follows: 
         [0015]    1. The bioreactor is developed to generate physiological pulsatile flow by mimic of the blood flow impedance, vascular compliance, and vascular inertia resistance in the flow loop. Pulsatile frequency, blood pressure, and flow waveform of different section of arterial  1  can be simulated in the bioreactor. The hemodynamic environment of high blood pressure, high sheer stress, and low sheer stress can also be simulated by adjusting the pulsatile waveforms, pressure, flow, and pulsatile frequency to a certain scope. 
         [0016]    2. The bioreactor is developed to impose controllable periodical axial tension, cyclical stretch and twisting, similar to the mechanical environment in vivo at the same time or separately. To impose the axial tension, at least one of the inlet tube and the outlet tube should be provided on the drive rod of the linear stepping motor to do axial movement reciprocal. 
         [0017]    3. The bioreactor is rotating-wall perfusion bioreactor for vascular construct. The vascular construct and the culture vessel can rotate at the same time or separately driven by rotary motor. The rotation speed and direction are controllable. The perfusion devices can perfuse culture media inside and outside the lumen at the same time or separately. So the bioreactor has a good mass transfer performance. 
         [0018]    4. The bioreactor could have mentioned a particular function, or any combination of a number of functions, or including all of the above-mentioned functions at the same time. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0019]      FIG. 1  shows a non-limiting embodiment of the present invention for illustrating the principle and arrangement of intravascular physiological pulsatile perfusion flow loop of the present invention. 
           [0020]      FIG. 2  shows a further non-limiting embodiment of the present invention, which comprises means for stretching vascular constructs to be cultured in the culture chamber. 
           [0021]      FIG. 3  shows a further non-limiting embodiment of the present invention, which comprises means for effecting extra-vascular perfusion of vascular constructs to be cultured. 
           [0022]      FIG. 4  shows a further non-limiting embodiment of the present invention, which allows for physiological pulsatile flow perfusion and rotation of the vascular construct and the culture chamber. 
           [0023]      FIG. 5  shows a further non-limiting embodiment of the present invention, which, in addition to the functions realized by the embodiment shown in  FIG. 4 , allows stretching of vascular constructs cultured in the culture chamber. 
       
    
    
     DESCRIPTION OF EMBODIMENTS 
       [0024]    Detailed description of embodiments of the present invention is given below with reference to drawings, in which like reference numerals denote same or similar parts, and some repetitive description thereof is omitted. 
         [0025]      FIG. 1  shows a non-limiting embodiment of the present invention for illustrating the principle and arrangement of the pulsatile flow generator of the present invention. As shown in  FIG. 1 , a pulsatile flow generator  301 , a first resistance adjustor  304 , and a first compliance chamber  305  are serially connected by liquid pipeline between an internal perfusion media reservoir  101  and a vascular constructs culture chamber  107 . 
         [0026]    As shown in  FIG. 1 , a pulsatile flow generator  301  of the present invention comprises a pulsation cavity  302 , an elastic soft tube  303  that goes through cavity  302 , an upstream one-way valve  308  provided at the upstream port of tube  303 , a downstream one-way valve  309  provided at the downstream port of tube  303 , a seal piston  310 , and a linear motion actuator  311  for driving piston  310 . Pulsation cavity  302  is a sealable cavity with a constant volume to be filled with liquid. Elastic soft tube  303  constitutes the part of the internal perfusion loop within pulsation cavity  302 . Elastic soft tube  303  is arranged in such a way that liquid within elastic soft tube  303  is separated from liquid filling cavity  302 , that is, there is no liquid exchange between the liquid filling cavity  302  and the liquid flowing through elastic soft tube  303 . On the other hand, the elasticity of the wall of tube  303  allows variation in pressure of the liquid filling cavity  302  to be transmitted to the liquid flowing through elastic soft tube  303 . Reciprocal movement of linear motion actuator  311  acts, by piston  310 , on the liquid filling cavity  302  and in turn on the culture liquid flowing through elastic soft tube  303 , thereby generating a corresponding pulsatile flow in the intravascular perfusion loop. 
         [0027]    As a preferred but non-limiting embodiment, such a pulsatile flow can be made to simulate the ejection of blood into aorta, etc., and the pulsatile frequency, flow rate, and/or pressure can be adjusted. One-way valves  308  and  309  ensure that the flow of culture liquid out of pulsatile flow generator  301  is unidirectional. 
         [0028]    Reference numeral  304  denotes a first resistance adjustor. A resistance adjustor is a mechanical adjusting device, such as an adjusting valve, provided on a section of pipe for adjusting the flow rate of liquid flowing through the pipe, which is accompanied by adjustment of perfusion pressure in the pipe. 
         [0029]    Reference numeral  305  denotes a first compliance chamber. A compliance chamber is for adjusting the variation in the liquid volume resulting from pressure variation. 
         [0030]    Reference numeral  306  denotes a second compliance chamber. Reference numeral  307  denotes a second resistance adjustor. 
         [0031]    Each of first and second resistance adjustors  304  and  307  is for adjusting perfusion pressure and waveform and/or amplitude of variation of perfusion pressure in the vascular construct  108 . Each of first and second compliance chambers  305  and  306  is for adjusting flow inertia of culture liquid in the vascular construct  108 . In an embodiment of the present invention, first and second resistance adjustors  304  and  307  and first and second compliance chambers  305  and  306  are used to obtain a physiological pulsatile flow, with its waveform, dicrotic wave, amplitude, and/or time phase, and/or to obtain the hemodynamic environment of high blood pressure, high sheer stress similar to hypertension, and to obtain the hemodynamic environment of low pressure, low sheer stress similar to hypotension. 
         [0032]      FIG. 2  shows a further non-limiting embodiment of the present invention. Comparing with the embodiment shown in  FIG. 1 , the embodiment of  FIG. 2  further comprises a section for stretching vascular construct being cultured in culture chamber. 
         [0033]    As shown in  FIG. 2 , reference numeral  105  denotes an upstream supporting frame of intravascular perfusion loop, reference numeral  104  denotes a culture chamber inlet pipe of intravascular perfusion loop. Reference numeral  110  denotes a culture chamber outlet pipe of intravascular perfusion loop. Reference numeral  702  denotes a driving rod of a stretching motor. Reference numeral  115  denotes a downstream supporting frame of intravascular perfusion loop. Reference numeral  701  denotes a stretch motor. 
         [0034]    As a non-limiting embodiment, culture chamber outlet pipe  110  fits with a downstream sealing plug  109  in a slidable way. The reciprocal stretching of driving  702  of stretch motor  701  acts on outlet pipe  110 , making outlet pipe  110  to perform axial reciprocal movement, thereby realizing reciprocal stretching of vascular construct  108  being cultured. 
         [0035]    Culture chamber  107  is preferably made with sterilization-tolerant material (such as glass, plastic, stainless steel, polycarbonate) to provide a sealed sterile environment for vascular construct to be cultured. During culture process of vascular construct, culture chamber  107  may be completely or partly filled with culture media; said culture media may be the same as the culture media flowing through the interior of vascular construct  108 . 
         [0036]    With an embodiment as shown in  FIG. 2 , axial reciprocal stretching of vascular construct  108  cultured and pulsatile flow perfusion in vascular construct  108  can be realized simultaneously. 
         [0037]    It should be understood that the arrangement of stretching motor  701  as shown in  FIG. 2  is not unique; and stretching motor  701  can be provided at the upstream side of culture chamber  107  and/or coupled to culture chamber inlet pipe  104  to obtain the same or equivalent effects. 
         [0038]    Further, stretching motor  701  is not the only way to effect reciprocal movement of vascular construct  108 , and it can be replaced by other devices, such as a crank-connecting rod mechanism, a hydraulic cylinder, or etc. 
         [0039]    All variations such as these are within the scope of the present invention. 
         [0040]      FIG. 3  shows a further non-limiting embodiment of the present invention, which, as compared with the embodiment of  FIG. 2 , further comprises parts for effecting extra-vascular perfusion of vascular construct  108 . 
         [0041]    The downstream end of culture chamber inlet pipe  104  of intravascular perfusion loop is provided inside culture chamber  107 . Reference numeral  601  denotes an optional upstream adaptor, which is connected to the downstream end of inlet pipe  104 . The upstream ends of a plurality of vascular constructs  108  to be cultured can be fitted on upstream adaptor  601 , thereby realizing simultaneous culturing of a plurality of vascular constructs. 
         [0042]    The downstream ends of vascular constructs  108  to be cultured can be fitted on a downstream adaptor  602 , which is connected to the upstream end of culture chamber outlet pipe  110  of the intravascular perfusion loop, while the downstream end of outlet pipe  110  is provided outside of culture chamber  107  and connects with a pipeline section leading to reservoir  101 , thus forming a closed intravascular perfusion loop. 
         [0043]    In  FIG. 3 , reference numeral  109  denotes sealing plugs for allowing inlet pipe  104  and outlet pipe  110  to enter into and/or to come out of culture chamber  107  respectively in a sealed way. 
         [0044]    A non-limiting embodiment as shown in  FIG. 3  further comprises an upstream supporting frame  105  and a downstream supporting frame  115  of intravascular perfusion loop; these frames are for supporting and/or holding inlet pipe  104  and outlet pipe  110 , respectively. 
         [0045]    An embodiment as shown in  FIG. 3  further comprises parts for collecting, processing, displaying and/or recording data; one embodiment as shown in  FIG. 3  comprises: a pressure sensor  201  provided at the inlet of culture chamber for sensing the pressure at the inlet of culture chamber  107  in intravascular perfusion loop; a stretching sensor  801  for detecting stretching force acted on inlet pipe  104 ; a displacement sensor  802  provided on the stretching motor for sensing a stretching amount of vascular construct  108 ; a hub  203  for receiving outputs of sensors  202  and  802 ; signal amplifier  204  for receiving outputs of sensors  202  and  802  from hub  203  and amplifying them; a driver  205 ; a processor  206 , which may be a PC or an IPC; and, a display  207 . 
         [0046]    As shown in  FIG. 3 , parts for effecting extra-vascular perfusion comprises: an extra-vascular perfusion media reservoir  501 , an extra-vascular perfusion liquid driving device  502  connecting to media reservoir  501  via liquid pipeline, an extra-vascular perfusion loop culture chamber inlet pipe  504 , which penetrates upstream sealing plug  109  and enters into culture chamber  107  for introducing culture liquid into culture chamber  107  from extra-vascular perfusion media reservoir  501 , and a culture chamber exit pipe  507  of extra-vascular perfusion loop, which penetrate a downstream sealing plug  109  for discharging culture liquid from culture chamber  107 . 
         [0047]    An embodiment as shown in  FIG. 3  can further comprises a culture chamber inlet pressure sensor  503  of extra-vascular perfusion loop and a culture chamber exit pressure sensor  508  of extra-vascular perfusion loop, for sensing liquid pressures at the inlet and exit of culture chamber of extra-vascular perfusion loop, respectively. Outputs of sensors  503  and  508  are sent to hub  203 , processed by processor  206 , and/or displayed by display  207 , etc. 
         [0048]    With an embodiment as shown in  FIG. 3 , extra-vascular perfusion in culture chamber is realized. 
         [0049]    With an embodiment as shown in  FIG. 3 , intravascular perfusion (physiological perfusion), extra-vascular perfusion, stretching of vascular construct(s), and any combination of these functions/effects can be realized simultaneously or separately. 
         [0050]      FIG. 4  shows a non-limiting embodiment of vascular construct bioreactor of the present invention. Details of such an embodiment are described below. 
         [0051]    As shown in  FIG. 4 , an intravascular perfusion media reservoir  101  is connected to a pulsatile flow generator  301  by a pipe section. 
         [0052]    The non-rotary pipe section at the downstream of pulsatile flow generator  301  is connected to the upstream end of rotary culture chamber inlet pipe  104  by an upstream coupling joint  103  of intravascular perfusion loop. Coupling joint  103  realizes a sealed connection between rotary inlet pipe  104  and the non-rotary pipeline leading to pulsatile flow generator  301 . 
         [0053]    The downstream end of inlet pipe  104  is provided inside culture chamber  107 . In a non-limiting embodiment as shown in  FIG. 4 , an upstream adaptor  601  connecting to the downstream end of inlet pipe  104  is provided, and a downstream adaptor  602  connecting to the upstream end of outlet pipe  110  is provided. The upper end of each of vascular constructs  108  to be cultured is fitted on upstream adaptor  601 . 
         [0054]    The downstream end of each of vascular constructs  108  is fitted on downstream adaptor  602 . The upstream end of outlet pipe  110  is provided inside culture chamber  107 , and the downstream end of outlet pipe  110  is provided outside of culture chamber  107  and connects, by a downstream coupling joint  112  of intravascular perfusion loop, to non-rotary pipeline leading to media reservoir  101 , thus forming a complete intravascular perfusion loop. Downstream coupling joint  112  effects a sealed connection between rotary outlet pipe  110  and the non-rotary pipeline leading to media reservoir  101 . 
         [0055]    As shown in  FIG. 4 , reference numerals  505  and  506  denote sealing plugs for allowing inlet pipe  104  and outlet pipe  110  to enter/exit culture chamber  107  respectively in a sealed manner. 
         [0056]    In a non-limiting embodiment as shown in  FIG. 4 , reference numeral  113  denotes a vascular construct rotation driving motor. Shaft  116  of motor  113  is coupled to an upstream transmission gear set  106  and a downstream transmission gear set  111 , so as to drive gear sets  106  and  111  to perform synchronized rotation. Gear set  106  is also coupled to inlet pipe  104 , and gear set  111  is also coupled to outlet pipe  110 , so rotation of gear set  106  drives inlet pipe  104  to rotate, and rotation of gear set  111  drives outlet pipe  110  to rotate, and the rotation of inlet pipe  104  is synchronized with the rotation of outlet pipe  110 , thus resulting in rotation of vascular construct(s)  108  provided between inlet pipe  104  and outlet pipe  110 . 
         [0057]    An embodiment as shown in  FIG. 4  further comprise parts for implementing independent rotation of culture chamber, which parts include a culture chamber rotary motor  604 , a culture chamber rotary transmission gear set  603  coupled to the shaft of motor  604 . Gear set  603  is further coupled to culture chamber  107  to transmit rotary driving force of motor  604  to culture chamber  107 . In a non-limiting embodiment of this coupling as shown in  FIG. 4 , a follower gear of gear set  603  is fixedly mounted on a collar  605  of culture chamber  107  to transmit the driving force of motor  604  to culture chamber  107 . In an embodiment as shown in  FIG. 4 , the joining between culture chamber  107  and sealing plugs  505  and  506  respectively is sealed and allows for relative rotation between culture chamber  107  and sealing plugs  505  and  506  respectively. 
         [0058]    With an embodiment as shown in  FIG. 4 , simultaneous and/or independent rotations of vascular construct and culture chamber can be implemented; in addition, separate rotation of vascular construct or culture chamber and/or different rotation combinations and rotation mode switching can be implemented. Therefore, more effective, uniform, and/or more effective media transfer can be provided to vascular construct(s) being cultured. 
         [0059]    It is to be noted that while two motors  113  and  604  are shown in  FIG. 4  for driving vascular construct and culture chamber respectively, the present invention is not limited to this. For example, a single motor with a clutch/transmission mechanism can be used to implement separate rotational driving of vascular construct(s) and culture chamber and/or various rotational driving modes. Such a modification is clearly within the scope of the present invention. 
         [0060]      FIG. 5  shows a further embodiment of the present invention, which, as compared with an embodiment as shown in  FIG. 4 , further comprises parts for effecting stretching of vascular construct being cultured. 
         [0061]    In an embodiment as shown in  FIG. 5 , culture chamber outlet pipe  110  fits with downstream sealing plug  506  in a slidable way. The reciprocal stretching of driving  702  of stretch motor  701  acts on outlet pipe  110 , driving outlet pipe  110  to perform axial reciprocal movement, thereby realizing reciprocal stretching of vascular construct  108  being cultured. 
         [0062]    Here, downstream transmission gear set  111  may accommodate its reciprocal axial movement relative to outlet pipe  110  in a variety of ways. 
         [0063]    A first way is that outlet pipe  110  is axially fixed with respect to the gear, which directly couples to outlet pipe  110 , of gear set  111 , and reciprocal axial movement of outlet pipe  110  is absorbed by axial sliding between gears of gear set  111 . for this, an optional arrangement is that one of the two gears in gear set  111 , between which sliding occurs, has a obviously greater thickness than that of the other one of the two gears, so that disengagement between the two gears due to sliding between them is avoided. 
         [0064]    A second way is that outlet pipe  110  is axially slidable with respect to the gear in gear set  111  that directly coupled to outlet pipe  110 , and a supporting brace (not shown) is used to axially fix the gear. 
         [0065]    With an embodiment as shown in  FIG. 5 , stretching of vascular constructs can be implemented in addition to internal and extra-vascular perfusion. 
         [0066]    It is to be noted that arrangement of stretching mechanism is symmetrical with respect to inlet pipe  104  and outlet pipe  110 , that is, the stretching drive of stretching motor  701  can either be coupled to outlet pipe  110  as shown in  FIG. 5  or be coupled to inlet pipe  104 . These two alternatives belong to the scope of the present invention. 
         [0067]    With an embodiment as shown in  FIG. 5 , rotation of vascular construct, rotation of culture chamber, perfusion (or physiological perfusion) inside and/or outside the lumen, stretching of vascular construct (s), and any combination of these functions/effects can be realized simultaneously or separately. 
         [0068]    It should be understood that gear sets  106 ,  111  and  603  are only exemplary for implementing corresponding rotary transmission devices. Other transmission mechanisms, such as chain transmission mechanism, belt transmission mechanism, rod transmission mechanism and etc., can be used to replace gear sets  106 ,  111 , and/or  603 . 
       Example 
     Intravascular and Extra-Vascular Perfusions with Vascular Construct Stretching, Vascular Construct Rotation and Culture Chamber Rotation 
       [0069]    1. Intravascular and extra-vascular perfusion loops were arranged as shown in  FIG. 5 , a pulsatile flow generator of the present invention was used as intravascular perfusion liquid driving device, and a peristaltic pump was used as extra-vascular perfusion liquid driving device (Cole-Parmer, Masterflex series); 
         [0070]    2. sterilization was performed on the bioreactor at 121□ (1 atm) for 1 hour; 
         [0071]    3. vascular constructs to be cultured were fitted to upstream and downstream adaptors in the culture chamber under aseptic conditions, the scaffold of the construct is 6 mm in diameter and 20 cm in length and made of PLGA. 
         [0072]    4. vascular construct rotation mechanism and culture chamber rotation mechanism were arranged as shown by  FIG. 5 , where the vascular construct rotation motor was a Haydon 57000 series linear step motor and the culture chamber rotation motor was a Haydon 57000 series linear step motor; 
         [0073]    5. pressure sensor at the inlet and outlet of culture chamber of intravascular and extra-vascular perfusion loops and signal detecting devices were arranged as shown by  FIG. 5 ; 
         [0074]    6. Vascular construct stretching device were arranged as shown by  FIG. 5 , where the stretching device comprised a Haydon 57000 series step motor; 
         [0075]    7. tension-compression sensor and displacement sensors were arranged as shown by  FIG. 5 ; 
         [0076]    8. culture medium was prepared as required; aseptic culture medium was filled into reservoirs; 
         [0077]    9. each of the devices was powered-on; 
         [0078]    10. pulsation frequency in lumen was set at 70 time/min., motor gain was set at 1-5%, and initial position was set; perfusion flow rate in lumen was set at 0-1.6 ml/s, inlet pressure was set at 100-140 mmHg, outlet pressure at 75-115 mmHg; perfusion flow rate outside the lumen was set at 0-1.0 ml/s, inlet pressure was set at 100-140 mmHg, outlet pressure at 85-110 mmHg; 
         [0079]    11. rotation speed and direction of the vascular construct rotation motor were set as: anti-clockwise, 10 rpm; rotation speed and direction of the culture chamber rotation motor were set as: clockwise, 20 rpm; 
         [0080]    12. periodic stretch stress on vascular constructs was set at 10N, and stretching frequency was set at 60 times/min.; 
         [0081]    13. operation of the bioreactor was started; 
         [0082]    14. resistance adjustors and compliance chambers were adjusted to control the pressure and waveform in vascular construct to simulate the artery pulse waveform similar to mammalian physiology.