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Prosthetic Grafts

a technology of prosthetic grafts and grafts, which is applied in the field of prosthetic grafts, can solve the problems of incomplete graft occlusion, synthetic or biosynthetic small vascular grafts, non-synthetic or biological, etc., and achieve the effects of enhancing angiogenesis, and enhancing patency in the prosthetic gra

Inactive Publication Date: 2007-04-19
ANDERSON DIANE LEE +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0056] According to the present invention, an adherent cell can also include a cell that has been genetically modified to be adherent, such the adherency becomes a natural characteristic of the genetically modified cell. A genetically modified cell is a cell that has been modified (i.e., mutated or changed) within its genome and / or by recombinant technology (i.e., genetic engineering) from its normal (i.e., wild-type or naturally occurring) form. For example, an endothelial cell is not considered to be an adherent cell according to the present invention, unless such cell has been genetically modified to be more adherent than a naturally occurring endothelial cell, in which case such a genetically modified endothelial cell is encompassed by the present invention. As discussed above, one advantage of the present invention over previously described prosthetic grafts is that the use of adherent cells on the outer surface of the graft eliminates the need for additional devices, delivery vehicles or binders that complicate preparation and compatibility of the graft and which can compromise the viability and stability of the recombinant cells and proteins produced by the cells.
[0057] Preferred adherent cells for use in the present invention include, but are not limited to fibroblasts, mesenchymal stem cells, bone marrow stem cells, embryonal stem cells, adipocytes, keratinocytes, vascular smooth muscle cells, platelets, and cells which have been genetically engineered to be adherent, with fibroblasts being particularly preferred.
[0058] The adherent cells of the present invention are transfected with at least one recombinant nucleic acid molecule that encodes one or more proteins that enhance patency in the prosthetic implant. Enhanced patency has been previously defined herein. According to the present invention, proteins that are particularly useful in enhancing patency in the prosthetic graft of the present invention include: a protein that enhances angiogenesis in the vascular bed downstream of the prosthetic graft, a protein that enhances angiogenesis transmurally and into the interior space of the prosthetic implant to endothelialize the inner surface of the prosthetic implant, a protein that inhibits thrombosis, a protein that causes thrombolysis, a protein that inhibits smooth muscle migration and / or proliferation, and / or a vasodilator protein.
[0059] Examples of proteins which are angiogenic (i.e., enhance or initiate angiogenesis) and / or are useful growth factors for enhancing patency include, but are not limited to: vascular endothelial growth factor (VEGF), platelet-induced growth factor (PIGF), transforming growth factor μ1 (TGFβ1), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), transforming growth factor α (TGFα), epidermal growth factor, osteonectin, angiopoietin 1 (Ang1), Ang2, insulin-like growth factor (IGF), platelet-derived growth factor AA (PDGF-AA), PDGF-AB and PDGF-BB. Examples of proteins which are useful for inhibiting thrombosis and / or causing thrombolysis include, but are not limited to: tissue plasminogen activator (TPA), streptokinase, hirudin V, αv-βIII, and urokinase plasminogen activator (uPA). An example of a protein which is useful for inhibiting smooth muscle cell migration and / or proliferation includes, but is not limited to nitric oxide synthase (NO synthase). An example of a vasodilator protein includes, but is not limited to prostacyclin. Other suitable proteins which can perform the above-described functions or otherwise enhance patency will be known to those of skill in the art. In addition, the amino acid and nucleic acid sequences for these proteins are known and therefore, the proteins can be readily produced recombinantly by a host cell using recombinant technology that is well known in the art.
[0060] According to the present invention, in one embodiment, all of the adherent cells which are adhered to the outer surface of the prosthetic implant can be transfected with the same recombinant nucleic acid molecule(s), so that each cell expresses the same recombinant protein(s). In another embodiment, adherent cells expressing different recombinant nucleic acid molecule(s) can be combined and adhered to the same implant. As such, several different proteins can be expressed on the same implant, and the proportions of the various proteins can be controlled by the proportion of adherent cells expressing each protein that are adhered to the outer surface of the implant or by the level of expression of the respective proteins.
[0061] Similarly, a single adherent cell is transfected with at least one recombinant nucleic acid molecule, but it is within the scope of the present invention that a single adherent cell can be transformed with two or more different recombinant nucleic acid molecules, so that a single adherent cell can express one, two, or multiple recombinant proteins. A single adherent cell can also be transfected with a single recombinant nucleic acid molecule that expresses one, two or multiple proteins, which can be under the control of the same transcription control sequence, or under the control of different transcription control sequences. In the case of expression of two or more recombinant nucleic acid molecules, the expression levels of the different molecules can be independently regulated, by, for example, controlling the copy number of the different recombinant nucleic acid molecules or by using different promoters to express the different proteins. According to the present invention, reference to “one” or “a single” recombinant nucleic acid molecule is intended to refer to one type of molecule or one particular sequence, but it is not to be interpreted to mean that a single host cell contains only one copy number of the molecule. For example, a host cell that is transfected with a single recombinant nucleic acid molecule can have and express one or multiple copies of the same recombinant nucleic acid molecule. It is to be noted that the term “a” or “an” entity generally refers to one or more of that entity; for example, a protein refers to one or more proteins, or to at least one protein. As such, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein. It is also to be noted that the terms “comprising”, “including”, and “having” can be used interchangeably.

Problems solved by technology

Research over the past several decades has yet to produce a synthetic or biosynthetic small bore vascular graft which can approach the patency rates of autologous vessels.
Platelet adhesion and activation at the lumenal surface of the graft are much more likely to result in complete graft occlusion.
Currently, non-synthetic or biological small bore grafts are routinely used as an arterial replacement since nothing has proven to perform nearly as well as the autologous saphenous vein or internal mammary artery, which are the conventional biological materials used as a small diameter vascular graft.
The harvesting surgery increases the total operating time and can also lead to complications and discomfort.
Furthermore, a small percentage of patients do not have autologous vessels suitable for, harvesting.
In some cases, the vessels are not available due to previous surgery, while in other cases, the vessel may be too small or varicose.
Even larger bore vessel and organ prosthetic grafts, however, suffer from complications associated with smooth muscle proliferation, compliance mismatch with native vessels, and poor endothelialization due to blood shear stresses and mechanical damage.
However, a completely non-fouling surface has yet to be discovered and many now view the quest for such a material as unrealistic.
Although a small number of grafts seeded lumenally with endothelial cells have been implanted clinically outside of the United States, and improved patencies over non-seeded grafts have been observed, this approach has generally enjoyed mixed success, and the concept still faces many challenges.
The harvesting surgical procedure not only increases prosthetic implant preparation time, but can also lead to complications and discomfort for the patient.
Second, retention of the cells on the graft surface after implantation has been an issue.
Although there is some evidence that methods such as conditioning may improve cell retention, all of these methods add yet another level of complexity to the seeding process and it is still not clear that significantly improved cellular retention can be achieved.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0097] The following example describes the production of a prosthetic graft according to the present invention.

Primary Cell Culture:

[0098] Primary Rabbit Aortic Fibroblasts (RAF) were obtained from young male White New Zealand rabbits. The aorta was explanted, the vessel was then longitudinally opened and the endothelium was removed by gently rubbing the lumenal surface with a scraper. After this step, the vessel was cut into small pieces, it was placed in a 60 mm dish containing 2 ml of Trypsin and kept at 37° C. in a 5% CO2 incubator for 60 minutes. After this incubation, the vessel was removed and placed in 60 mm dishes with 0.5 ml of DMEM supplemented with 10% FBS containing 2 mM L-glutamine and 100 UI / mL Pen / Strep. After overnight incubation, 1.5 ml of culture medium was added. RAFs were cultured with DMEM with 10% FBS, 2 mM L-glutamine and 100 UI / mL Pen / Strep in a humidified 5% CO2 atmosphere at 37° C. Proliferating cells were used between passage 3 and 8 and were used for ...

example 2

[0103] The following example demonstrates that a prosthetic graft of the present invention produces the recombinant protein both inside and outside of the graft under static in vitro culture conditions.

[0104] Production and secretion of VEGF under static in vitro conditions was measured from the PhotoFix graft seeded perivascularly with rabbit aortic fibroblasts infected with AdV cmv VEGF as described in Example 1. Briefly, the seeded PhotoFix was mounted within a closed circuit placed in a chamber and connected to a peristaltic pump. Five ml of medium in static condition were placed inside the PhotoFix, while 300 ml of medium was placed in the incubation chamber outside the PhotoFix. The chamber was incubated under static conditions under shear stress in a humidified 5% CO2 atmosphere at 37° C. At the end of incubation, external and internal medium was recovered (the internal medium was collected through a plastic outlet mounted on the circuit) and stored at −20° C. for the ELISA ...

example 3

[0107] The following example demonstrates that a prosthetic graft of the present invention produces the recombinant protein both inside and outside of the graft under shear stress in vitro culture conditions.

[0108] Release of VEGF under dynamic condition-shear stress of 1.5 dyn / cm2 during in vitro conditions was measured from the PhotoFix graft seeded perivascularly with rabbit aortic fibroblasts infected with AdV cmv VEGF as described in Example 1. Briefly, as described in Example 2 above, the seeded graft was mounted within a closed circuit placed in a chamber and connected to a peristaltic pump. For the dynamic condition assays, 15 ml of medium were placed inside the PhotoFix and 300 ml of medium was placed in the incubation chamber outside the PhotoFix. The chamber was incubated in a humidified 5% CO2 atmosphere at 37° C. under dynamic conditions of shear stress by circulating the medium through the circuit at a velocity of 1.5 dyn / cm2 as indicated. The medium was collected and...

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Abstract

An improved prosthetic graft for the bypass, replacement or repair of vessels and organs that are in contact with blood flow is disclosed. The prosthetic graft includes a porous prosthetic implant and adherent cells adhered to the outer surface of the implant. The adherent cells are transfected with at least one recombinant nucleic acid molecule encoding at least one protein that enhances patency of the graft. The prosthetic graft has a long-term patency and success rate that is superior to other previously described prosthetic grafts designed for such use. Also disclosed are methods of making and using such a graft.

Description

FIELD OF THE INVENTION [0001] The present invention relates to prosthetic grafts which are used to contain blood flow in vivo. BACKGROUND OF THE INVENTION [0002] Diseases of the major circulatory and renal organs and vessels have created a need for prosthetic grafts to bypass, repair and / or replace the function of the diseased organs and vessels. Such grafts should ideally be non-immunogenic, non-calcific, and readily capable of recreating or reestablishing the natural blood contact interface of the organ or vessel to be replaced or repaired. Complications that have inhibited the widespread use of prosthetic grafts in organs and vessels in contact with blood include: (1) intimal hyperplasia, whereby smooth muscle cell and myofibroblast proliferation and extracellular matrix accumulation cause thickening of the intima in the graft and in the adjoining vessels, and ultimately lead to failure of the graft; and, (2) occlusion of the graft, whereby platelet adhesion and activation at the...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01N63/00A61K48/00A61F2/06A61K35/12A61L27/38A61L27/50C12N5/02C12N5/077
CPCA61F2/06A61F2/062A61K48/00A61K48/0075A61K2035/126A61L27/38A61L27/3804A61L27/3843A61L27/507C07K14/52C12N5/0656C12N2510/02C12N2799/022C12N2799/04
Inventor ANDERSON, DIANE LEERANIERI, JOHN PAULCOLOGNESI, MAURIZIO CAPOGROSSISCOCCIANTI, MARCOFACCHIANO, ANTONIO
Owner ANDERSON DIANE LEE
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