Patent Application
20090123993
1. Field of the Invention
The present invention relates to bioreactor systems for vascular graft culture and tissue engineering. More particularly, the present invention relates to a computer-controlled bioreactor system useful for harvesting, transporting, long-term maintenance and development of blood vessels for cardiovascular disease investigation, drug testing and tissue engineering.
2. Description of Related Art
Cardiovascular disease (CVD) is a leading cause of death in the United States. Over 2,600 people or one person every 30 seconds dies from heart disease each day. The cost (direct and indirect) of cardiovascular diseases and stroke in 2004 was estimated to be $368.4 billion in the United States, according to the American Heart Association and the National Heart, Lung, and Blood Institute. The World Health Organization estimates that 16.9 million people around the globe die of cardiovascular diseases each year.
Individuals who are genetically predisposed to CVD, such as diabetics, and those individuals who develop atherosclerosis, require vessel clearance, repair, or replacement. Vessel clearance is accomplished by routing the obstructed channel until a degree of patency is obtained. Vessel repair can be manifested by fragmenting atherosclerotic plaque or other obstructions by vessel expansion using a balloon catheter, then inserting a stent to maintain vessel patency.
Large vessels can readily be treated or repaired. For example, large vessels that have undergone pathologic change, such as a dissecting aneurysm or burst wall, and thus which require partial replacement, may be replaced with DACRON™ or GoreTex™ tubular devices which are sutured to either end of the damaged vessel. In addition, rather than surgically remove a segment of a damaged blood vessel, the damaged section of the vessel can be treated with an internal sleeve. In this way, the walls of the original vessel are left intact and the damaged internal wall is repaired with an internal stent that prevents further tearing and dehiscence of the intima, or endothelial lining of the vessel. In contrast, treatment and repair of smaller vessels have proven more problematic. For example, challenges remain for the repair or replacement of mid-size arteries that service the heart muscle, such as the carotid arteries. These vessels have approximately a 3-5 mm internal diameter bore size and represent a major portion of the surgical cases that usually can be repaired only by vessel expansion and stent placement. A major problem is that 50-60% of these vessels occlude again after repair and stent placement within six to twelve months, even when an anti-clotting medication, such as coumadin, is administered. Additionally, smaller vessels cannot be replaced with synthetic materials, such as DACRON™ or GoreTex™, because these materials cause clotting in smaller bore tubes in vivo.
Large vessel synthetic graft devices have been available for many years. However, engineering small diameter vascular devices or tissue engineered constructs has proven more difficult because of the increased incidence of thrombosis and occlusion which typically occur with these devices and constructs. Artificial vessel endothelialization is one of the methods used to solve the challenge of thrombosis (blood clotting). However, human endothelial cells adhere weakly to currently available vascular graft materials. Some expanded polytetrafluoroethylene grafts have shown only about a 10%+/−7% overall attachment of endothelial cells. Moreover, when a graft is exposed to pulsatile blood flow, a higher proportion of cells may elute from the luminal wall, with maximum cell detachment occurring in the first 30-45 minutes after exposure to pulsatile flow and with up to 70% of the initial cell population removed during that time; a lower rate of detachment occurring over the next 24 hours. The lack of cell retention may be overcome partly by additional cell seeding. Other techniques, such as engineering the vessel lumen with adhesion factors, have been developed to improve endothelial cell adhesion and retention rate. These techniques include shear stress preconditioning, electrostatic charging and precoating the lumen with endothelial cell-specific adhesion glues that are characteristic of the extracellular basement membrane of blood vessels. The most common techniques are chemical coatings, preclotting, chemical bonding and surface modifications (Xiao, Chin., J. Traumatol., 7(5):312-6, 2004). Additionally, substantial efforts are being invested by the bioengineering community to develop biodegradable polymer scaffolds suitable for tissue engineering applications. Many new materials have been developed such as a poly (L-lactide-co-epsilon-caprolactone) copolymer, which has a novel architecture produced by an electrospinning process.
Biodegradable materials formed in tubular shapes seeded with autologous cells have attracted much interest as potential cardiovascular grafts. However, pretreatment of these materials with cells requires a complicated and invasive procedure to collect vessel tissue, culture the cells and seed the graft before implantation in a patient. This procedure requires two surgical procedures and carries a risk of infection. A biodegradable graft material which contains a collagen microsponge that encourages the regeneration of autologous vessel tissue has been developed which appears to overcome these problems. The poly (lactic-co-glycolic acid)-collagen microsponge patch with and without precellularization has shown to have good histologic properties and durability (Iwai S., J. Thorac. Cardiovasc. Surg., 128(3):472-9, 2004).
However, the success of tissue engineering and biomaterial applications in blood vessel fabrication not only is dependent on the initial functioning of the device with regard to patency, anastomosis and bursting strength, but also is dependent on the successful vascularization of the implant itself after implantation. The process of vascularization involves angiogenesis, i.e., the formation of new blood vessels which spread into the implant material and supply the existing cells with the nutrients to survive. In vitro methods have been established using human microvascular endothelial cells to populate novel biomaterials to test endothelial cell attachment, cytotoxicity, growth, angiogenesis and the effects on gene regulation. Results from in vitro studies may be used to evaluate the potential success of a new biomaterial and for the development of matrix scaffolds which will promote a physiological vascularization response (Kirkpatrick, C. J., J. Mater. Sci. Mater. Med., 14(8):677-81, 2003).
Currently, biomaterials are widely used in medical sciences. The field of biomaterials began to shift to produce materials able to stimulate specific cellular responses at the molecular level. The combined efforts of cell biologists, engineers, materials scientists, mathematicians, geneticists, and clinicians now are used in tissue engineering to restore, maintain, or improve tissue functions or organs. This rapidly expanding approach combines the fields of material sciences and cell biology for the molecular design of polymeric scaffolds with appropriate three-dimensional configuration and biological responses.
Tissue engineering of biomaterials may offer patients new options when replacement or repair of an organ is needed. However, most tissues require a microvascular network to supply oxygen and nutrients. One strategy for creating a microvascular network is to promote vasculogenesis in situ by seeding vascular progenitor cells within the biopolymeric construct. To pursue this strategy, CD34(+)/CD133(+) endothelial progenitor cells (EPCs) have been isolated from human umbilical cord blood and expanded ex vivo as EPC-derived endothelial cells. EPCs appear to be well suited for creating microvascular networks within tissue-engineered constructs (Bischoff, J., Am. J. Physiol. Heart Circ. Physiol., August; 287, 2004). Additionally, basic fibroblast growth factor (bFGF) coating has been tested to promote endothelial cell seeding and proliferation on a decellularized heparin-coated vascular graft. This coating has been shown to increase the retention of seeded cells on the graft under flow conditions. In one study, after only three hours of cell attachment, 60% of human microvascular endothelial cells (HMECs), as well as canine peripheral blood endothelial progenitor cells (CEPCs), were shown to be retained in intact grafts exposed to flow relative to the static control graft group, thus demonstrating that bFGF coating on the heparin bound decellularized grafts significantly increased both HMEC and CEPC proliferation and that seeded cells remained stable under perfusion conditions (Conklin, B. S., Artif. Organs., 28(7):668-75, 2004).
Autologous transplantation of “artificial blood vessels” as arterial interposition grafts has been performed successfully in a canine model, in which peritoneal and pleural cavities of large animals have been shown to function as bioreactors to grow myofibroblast tubes for use as autologous vascular grafts (Chue, W. L., J. Vasc. Surg., 39[4]:859-67, 2004). Progress in tissue engineering now allows the recreation of functional blood vessels from cultured human vascular cells. When reconstructed under specific conditions, their structure, mechanical properties and function (especially vasomotricity) allow them to be used as human models for studying the biology and pharmacology of blood vessels.
To date, biomedical investigators have developed methods to remove pathologic blood vessels from patients in procedures that involve coronary or carotid artery opening, vena cava removing or sleeving and vein stripping. Surgical techniques are well developed that result in a vessel that can be anastomosed by suturing the implant ends to existing ends of blood vessels. Initially, investigators have focused on the use of autologous blood vessel parts to repair or replace vessels damaged by trauma or disease. Materials scientists have joined with clinicians to develop synthetic devices, such as the DACRON™ vena cava replacement. Implantation of this device requires the removal of the enlarged, pathologic vessel segments and the sutured connection of the radiator hose-like device to the living vessel ends. Similar devices comprised of GoreTex™ have been implanted in a similar fashion. There are principle concerns with the use of these devices, however, which include the strength of the anastomoses, as the connection of the device is affected by suturing to the living vessel ends; and leakage of blood through the permeable textile wall and subsequent blood clotting on the outside wall as well as the inside wall. An initial design feature had included blood clotting itself, which sealed the device from further leakage of blood into the body cavity. However, patients had to be treated with anticlotting agents, such as coumadin, to prevent excessive clotting and a possible life-threatening embolism that could occlude a vessel downstream from the device.
Research and development have continued on the design of novel biomaterials and architectures for vessel replacement devices that are synthetic, biosynthetic or biological. For biological devices, a bioreactor typically is required to sustain cell viability and stimulate development of cells to maintain and build a vessel architecture which functions without clotting, bursting or leakage at the anastomoses or through the vessel wall. This level of design, fabrication and use that results in the development of a substitute vessel that acts sufficiently well to replace an existing blood vessel or other conduit in the body is referred to as Functional Tissue Engineering of a substitute vessel. Bioreactor designs suitable for the maintenance or development of blood vessels involve at least two major aspects: (1) the development of a tubular scaffold that can be used as a framework for vessel-specific cell attachment and function; and (2) the design of a bioreactor that provides a suitable environment in which to maintain and/or develop a blood vessel phenotype for the cells which may be seeded into the scaffold lumen, interior wall and exterior wall. The engineered blood vessel substitute may also have specially designed ends which provide functional attachment points from the engineered vessel to the ends of the living vessels for anastomosis.
Examples of tubular scaffold designs include, but are not limited to, extruded materials, sheets of materials that are rolled into a tube and contain a seam, sheets of materials that are rolled into a tube and are seamless, sheets of materials that form an undulating pattern and are mated with a mirror image-shaped material to form tubes, undulating sheets of material that have a flat sheet of a secondary material bonded to the surface to form tubes, materials that are formed on a mandril into a tube, materials that are spun into a tube, materials that are woven into a tube, rings of material that are stacked to form a tube, solid materials that have material removed so that a cavity or cavities remains which are laser or otherwise “burned” to create a tube or tubes, particles that are progressively laid down in a pattern to form a tube, materials that are expanded to form a tube, and materials that condense to form a tube. Currently, no dominant type of tube formation has prevailed. However, typically a blood vessel open pore scaffold is obtained by providing a tubular shape formed by extrusion or seamless sheet folding into a tube in which the tube is populated with endothelial cells on the inner (luminal) surface and with smooth muscle cells in the interstices of the tube wall. Adventitial cells then can be seeded on the exterior surface of the scaffold. In this way, the gross anatomy of the blood vessel with respect to major histologically defined layers is simulated.
Bioreactors have also been developed to maintain blood vessels removed from the body or to stimulate the development of bioengineered vessels. Most of the bioreactor designs involve a single or multiple of cylindrical chambers with capped and plugged inlet and outlet ends that service fluid flow through the blood vessel lumen. A second inlet and outlet service the chamber surrounding the exterior wall of the blood vessel. Hence, the design typically includes a tube within a tube chamber in which the inner tube is the blood vessel with its flow independent of the flow of the exterior chamber which provides the nutrient flow to the blood vessel exterior. Each flow path is controlled by a syringe pump or peristaltic pump with pressure controllers, pulse dampeners and regulation of flow rate. Certain blood vessel bioreactors also provide uniaxial strain to simulate strain experienced by a vessel upon elongation of the draining limb, or torque to simulate the twisting motion of, for example, limb muscles. However, no evidence has been reported that these added mechanical features actually benefit the blood vessel or bioengineered construct to increase their mechanical strength or to maintain a blood vessel phenotype. Indeed, there usually is some difficulty in clamping or suturing a blood vessel segment onto an inlet or outlet fixture and loading the blood vessel into a cylindrical tube. Furthermore, there also is the problem of maintaining sterility for long periods in conventional bioreactors, particularly if sampling from the medium is routinely performed.
There exists a need, therefore, for a blood vessel bioreactor system which can harvest, transport, maintain and develop native or biosynthetic blood vessels from small bore to large bore sizes under aseptic conditions.
The present invention provides a blood vessel bioreactor and accessory system which can be used to maintain a native blood vessel or to develop a tissue-engineered biosynthetic blood vessel construct in which regulated nutritive fluid flow as well as a pressure and shear stress regimen is supplied which nutritionally and mechanically conditions the native or tissue-engineered blood vessel construct to functionally withstand an in vitro or in vivo environment. Additionally, engineered parts of the bioreactor system are designed to encourage ingrowth of the vessel ends into functional integrative connections which can readily be joined to a living blood vessel tissue in the body.
In particular, the blood vessel bioreactor and accessory system of the present invention is a complete system which allows for the stepwise isolation of a blood vessel segment in situ, engagement of a defined length and bore size of the blood vessel segment with a blood vessel attachment/engagement cuff, severing of the blood vessel segment, attachment of the severed ends of the blood vessel segment with various types of blood vessel inlet and outlet cuff connectors that provide a positive connection to a nutritive fluid flow system, and a cassette-type bioreactor cartridge for the harvesting, transport, maintenance and/or development of native or tissue-engineered blood vessels. The novel features of the blood vessel bioreactor system allow for ease of collection of a living blood vessel in an animal or human for the purpose of removal of the blood vessel and connection to other blood vessels in the body and/or connection to a bioreactor flow system for the purpose of conditioning the blood vessel(s) by biochemical and/or electrical and/or mechanical means.
The blood vessel bioreactor system therefore is capable of engaging a blood vessel segment on its exterior surface by vacuum in a defined way and geometry for the purpose of gauging its length, engaging it in a manner that does not excessively compress or damage the blood vessel ends and allows for the defined severing of the blood vessel so that it can be removed from the body of an animal or human. The blood vessel bioreactor system includes a blood vessel transport cassette for the transport of the blood vessel segment from one site, such as an operatory or surgical suite, to another site for the purpose of reimplantation, maintenance and/or culture of the blood vessel. The connections in the bioreactor which supply the blood vessel with nutrient fluid flow are designed to allow ease of connection and disconnection from the bioreactor unit and subsequent implantation in a patient or other subject.
The present invention provides a novel blood vessel bioreactor system for the harvest, maintenance, transport and/or development of native blood vessels or other tubular biological structures, such as tissue-engineered biosynthetic blood vessel constructs. The bioreactor system is comprised of a blood vessel harvest and carriage cassette and a computer-operated blood vessel bioreactor flow system for the harvest, maintenance, transport and/or development of native blood vessels or other tubular biological structures, such as tissue-engineered biosynthetic blood vessel constructs.
A complete understanding of the present invention will be obtained from the following description taken in connection with the accompanying drawing figures, wherein like reference characters identify like parts throughout.
In an embodiment of the present invention, as shown in
The interior of the tubing 42 contains a nutritive fluid flow to provide nutritive fluid through the blood vessel inlet/outlet connector cuff 40 to the interior of the blood vessel segment 22. Additionally, the blood vessel bioreactor 30 has a plurality of knobs 44 located on the external surface of the bioreactor 30 which connect to the tubing 42 to adjust the length of the tubing 42 in conformance with the length of the blood vessel segment 22.
Each end of the blood vessel inlet/outlet connector cuff 30 also is configured to connect to adjustable, rigid tubing 42 having a nutritive fluid flow therein to provide nutritive fluid to the exterior of the blood vessel segment 22.
The bioreactor 30 optionally has a transparent cover 34 and locking clamps 36 for sealing the transparent cover 34 onto the bioreactor 30.
As shown in
In an embodiment of the present invention, the blood vessel bioreactor system is equipped with sensors capable of providing a read-out of O2 and CO2 content, pH, pressure and bacterial contamination (assessed by, for example and without limitation, turbidity or conductivity of the fluid medium) in the internal fluid medium as well as in the external fluid medium. Additionally, the computer-operated flow control system is able to regulate the flow within the lumen of the tubings, blood vessel inlet and outlet cuff connectors and blood vessel segment with respect to duration, flow rate and directionality so as to provide a regulated steady flow, an oscillating flow, or flow reversals, as provided in the commercially available STREAMER™ flow device manufactured by Flexcell International Corp. Additionally, the blood vessel bioreactor system of the present invention is configured to apply regulated, uniaxial tension to the blood vessel segment by means of one or both ends of the blood vessel inlet and outlet cuff connectors which translate axially according to a computer-generated programmable regimen.
In another embodiment of the present invention, the bioreactor system can be set up as a series of bioreactors which are engaged on a linear or circular frame with separate or shared nutritive fluid flow systems.
It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications that are within the spirit and scope of the invention, as defined by the appended claims.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US06/12086 | 3/30/2006 | WO | 00 | 6/18/2008 |
| Number | Date | Country | |
|---|---|---|---|
| 60667489 | Mar 2005 | US |
