ES2348961T3 - COMPOSITIONS AND THEIR USE TO INCREASE VASCULAR ACCESS. - Google Patents

Abstract

An implantable material comprising cells and a biocompatible matrix for use in the treatment of an arteriovenous fistula, an arteriovenous graft or a peripheral bypass graft in a patient, comprising the step of locating the implantable material on an outer surface of the fistula or graft, in which the implantable material is effective in promoting a safe remodeling of the fistula or graft, thereby effecting vascular dilation with simultaneous inhibition of hyperplasia of the neointimal venous layer.

Description

Background of the invention

The failure of vascular access is the main complication in the provision of assistance to hemodialysis patients to treat end-stage renal failure (hereinafter ESRD). The rate of current ESRD cases in the United States has increased every year since 1980. In 2001, the rate of cases already diagnosed reached almost 1,400 patients per million inhabitants, representing an increase of 2.4% over to the previous year Based on demographic changes in age, race, ethnicity and diabetic status, the population of ERSD already diagnosed in the United States is expected to grow to 1.3 million by 2030. Currently, approximately 65% of the population With ERS already diagnosed, he is treated with hemodialysis (approximately 264,710 patients). Between 1,997 and 2,001, the population with hemodialysis already diagnosed grew by 4.5% per year. Using data from the Medicare program, it has been estimated that by 2001 the total costs of ERSD reached 15.5 billion dollars, which represented 6.4% of the entire budget of the Medicare program of 242,000 million (total costs reached $ 22.8 billion of all items). In fact, the annual cost of morbidity related to vascular access in the United States currently exceeds $ 1 billion annually.

Vascular access failure is the most important individual cause of morbidity in the hemodialysis population. A recent communication that analyzed the data of the renal data system of the United States (hereinafter USRDS) obtained a global primary patent rate of unassisted access of only 53% in 1 year. The rates of primary patents for non-assisted access in 1 year were 49% for vascular access structures such as arteriovenous grafts involving prosthetic bridges of expanded polytetrafluoroethylene (hereinafter ePTFE) and 62% for arteriovenous fistulas. (hereinafter AV). Accumulated patent rates for first-time accesses in 1.3 and 5 years were 54%, 46% and 36% for forearm fistulas and 54%, 28% and 0% for AV grafts, respectively. Currently, the use of grafts involving prosthetic ePTFE bridges amounts to 70% of all hemodialysis access interventions in the United States; Currently, the National Kidney Foundation recommends that AV fistula be the preferred method of vascular access. It is expected that in the future there will be an increase in the proportion of new AV fistulas in the United States.

Arteriovenous autofistulas have historically been considered as the best option for vascular access in hemodialysis patients. When an AV fistula matures satisfactorily after a surgical creation, it could work for years with a low risk of complications and a small incidence of revisions. However, the reported rates of AV fistula immaturation vary widely, but continue to be approximately 2050%. Immaturation is generally defined as the inability to allow repetitive cannulation of the fistula for dialysis or to obtain sufficient blood flow for dialysis within 12 weeks after surgical creation. The occurrence of immaturation of AV fistulas may depend, in part, on the quality and size of the vessels used to form the AV fistula. It has been shown that preoperative evaluation of vessel characteristics has beneficial effects in identifying suitable vessels for the creation of AV fistulas.

The failure of vascular access structures is attributable to the cumulative effect of various acute and chronic phenomena, especially in the so-called "toe" and its surroundings running forward. For example, AV grafts could develop stenosis in relation to association with the graft and occlusions in relation to association with the graft in anastomoses on the venous anastomosic side. In a published communication, histological examination of segments extracted from patients with anastomotic stenosis in relation to association with a graft revealed an intimal layer hyperplasia consisting of smooth muscle cells and extracellular matrix. Graft thrombosis may also contribute to vascular access dysfunction in ePTFE dialysis grafts. In addition, in general the isolation of veins and arteries followed by exposure of the vein segment to arterial blood flow and pressure can cause inevitable ischemia and revascularization injury. Surgical manipulation and suturing can also result in direct trauma to the endothelium and smooth muscle cells of the fluids in both veins and arteries. Injury to the artery and vein endothelium during the creation of a native or graft anastomosis can influence the patency and occlusion rates. In addition to physical trauma in relation to association with the cutting and suturing of veins and arteries during the formation of a vascular access structure, an increase in wall tension and shear force can also cause physical and / or biochemical lesions. to the endothelium It has been suggested that blood pressure could alter the normal production of endothelial growth regulating compounds, as well as produce morphological and biochemical changes in vein fluid.

Current therapy for vascular access failure is either surgical revision or angioplasty with or without stents. Surgical treatment could be risky in these typically multipathological patients and the long-term results of angioplasty and stents are generally disappointing due to the failure rates of their own. Therefore, the objective of a perfected vascular access for hemodialysis purposes as well as for peripheral circulation is to maintain the anatomical integrity of the original graft area to allow blood flow rates to support dialysis treatment or sufficient blood flow in areas of peripheral shunt.

Other factors that contribute to the satisfactory maturation of a newly created vascular access structure or the prolonged maturation of an existing vascular access structure remain difficult to find. In addition, relatively few randomized clinical trials have been carried out in the field of preventing vascular access failures. Studies that have evaluated the causes of vascular access failure have reached inconsequential conclusions. In fact, at the present time, despite the enormity of this problem, doctors do not have surgical, therapeutic or pharmacological measures for the prolonged survival of functioning fistulas. Obviously, there is a need to step forward in this vital area of patient care.

US 6328762 describes implantable grafts that take the form of porous tubular prostheses in which the outer surface is coated with adherent cells. This document does not describe the placement of a cell matrix implant on an outer surface of a vessel to be treated, nor the use of the implant to effect a positive remodeling of a fistula or graft.

US4820626 describes implantable tubular prostheses in which the contact surface is seeded or coated with endothelial cells for use as vascular grafts. The grafts do not make contact with an outer surface of a blood vessel, but instead are surgically grafted (therefore integrated) into the diseased vasculature in order to maintain luminal continuity.

Summary of the invention

The present invention exploits the discovery that an implantable material comprising cells and a biocompatible matrix, when provided locally to a vascular access structure, can promote the formation or increase the maturation of the structure, as well as prolong the structure in a state mature functional. In accordance with the present invention, an implantable material is provided as defined in the claims. The present invention can effectively promote the integration or increase the maturation of a newly created vascular access structure, promote and sustain the functional duration of a functioning current structure, and can help in the recovery of a damaged or spoiled structure.

In the case of an arteriovenous natural fistula, the treatment of the natural arteriovenous fiber increases the clinical maturation sufficient to allow hemodialysis, reduces the delay in the maturation of the natural arteriovenous fistula and promotes repetitive cannulation. In the case of an arteriovenous graft, the treatment of the arteriovenous graft promotes sufficient clinical stability to recover normal or near normal circulation. In several of the embodiments, the implantable material reduces the frequency of revision in a patient having an access structure.

Increased maturation is characterized by an ability to repeatedly cannulate the fistula for dialysis or by an ability to obtain sufficient blood flow during dialysis. Preferably, a sufficient blood stream comprises a flow rate of approximately 350 ml / minute. The application of the biocompatible material to the arteriovenous fistula could be preceded by - or be coincident with - a therapeutic agent, a physical dilation or a stent. The arteriovenous fistula could be radiocephalic, brachiocephalic or brachiobasilic.

In another aspect, the invention is an implantable material comprising cells and a biocompatible matrix suitable for treating a vascular access structure. Cells are endothelial cells or cells that have an endothelial-looking phenotype. The biocompatible matrix is a flexible flat form or a flowable composition. In a particularly preferred embodiment, the cells are vascular endothelial cells. The flexible flat shape is configured for implantation in, next to, in the vicinity of a native fistula ... According to one embodiment, the biocompatible matrix is a composition that retains the shape.

In other embodiments, the biocompatible matrix is a flexible flat form or a flowable composition. In one embodiment, the flexible flat shape is configured for implantation in, next to, or in the vicinity of a native fistula. In certain embodiments, this form is configured such that it defines a slot or a series of slots. With respect to the flowable composition, it could be a composition that retains the shape.

Brief description of the drawings

In the drawings, similar reference numbers refer to the same parts throughout all the different views. Also, the drawings are not necessarily to scale or in proportion, with the emphasis being placed instead on the illustration of the principles of the invention.

Figure 1 is a schematic perspective view of a flexible flat form of implantable material for administration to an outer surface of a tubular anatomical structure according to an illustrative embodiment of the invention.

Figure 2A is a schematic perspective view of a flexible contoured flat shape of implantable material for administration to an outer surface of a tubular anatomical structure according to an illustrative embodiment of the invention.

Figures 2B, 2C, 2D, 2E, 2F and 2G are schematic perspective views of a contoured flexible flat shape of the implantable material comprising a groove according to various illustrative embodiments of the invention.

Figures 3A and 3B are representative cell growth curves according to an illustrative embodiment of the invention.

Figures 4A, 4B and 4C illustrate a series of steps for administering multiple flexible flat presentations of implantable material to an outer surface of a vascular anastomosis from a perspective view from above according to an illustrative embodiment of the invention.

Figure 5 is a perspective view from above of a contoured form of implantable material administered to an outer surface of a vascular anastomosis according to an illustrative embodiment of the invention.

Figure 6 is a perspective view from above of a flexible flat form of implantable material administered to a tubular anatomical structure according to an illustrative embodiment of the invention.

Detailed Description of the Invention

As explained herein, the invention is based on the discovery that a cell-based therapy can be used to treat vascular access structures. The teachings presented below provide sufficient guidance to construct and use the materials of the present invention, and also provide sufficient guidance to identify suitable criteria and topics for testing, measuring, and monitoring the performance of the materials and methods of the present invention.

In accordance with the above, a cell-based therapy has been developed to clinically manage vascular access complications and / or failures. An exemplary embodiment of the present invention comprises a biocompatible matrix and cells suitable for use with the treatment paradigms described herein. Specifically, in a preferred embodiment, the implantable material comprises a biocompatible matrix and endothelial cells or an endothelial cell-like cells. In one embodiment, the implantable material is in a flexible flat form and comprises endothelial cells or endothelial-like cells, preferably human aortic endothelial cells and the Gelfoam® gelatin sponge of biocompatible material (manufactured by Pfizer, New York, NY, of hereinafter "Gelfoam matrix"). According to another preferred embodiment, the implantable material is in a form that can flow and comprises endothelial cells or endothelial-like cells, preferably human aortic endothelial cells and Gelfoam® gelatin particles or powder (manufactured by Pfizer, New York) (in forward "Gelfoam particles") as a biocompatible matrix.

The implantable material of the present invention comprises grafted cells on, in or within a biocompatible matrix. The term "grafted" means firmly fixed by means of cell-to-cell or cell-to-matrix interactions so that the cells withstand the rigors of the preparatory manipulations described herein. As explained elsewhere in this specification, an operative embodiment of implantable material comprises a population of almost confluent, confluent or post-confluent cells having a preferred phenotype. It is understood that embodiments of implantable material will likely shed cells during preparatory manipulations and / or that certain cells are not fixed as firmly as other cells are. All that is required is that the implantable material comprises cells that meet the functional or phenotypic criteria specified herein.

The implantable material of the present invention was developed based on the principles of tissue engineering and represents a novel solution to meet the clinical needs outlined above. The implantable material of the present invention is unique in the sense that viable cells grafted onto, in or within the biocompatible matrix are capable of supplying multiple cell-based products in physiological proportions under control to the vascular access structure and corresponding vasculature. of physiological feedback. As described elsewhere in the present specification, cells suitable for use with the implantable material are endothelial or endothelial-like cells. The local administration of multiple compounds through these cells and a physiologically dynamic dosage provide a more effective regulation of the processes responsible for maintaining a functional structure of vascular access and reducing complications or failure of vascular access. It is an important fact that the endothelial cells, for example, of the implantable material of the present invention are protected from erosive blood flow within the lumen of the vessel, due to their location on a non-luminal surface of the vessel, for example, in the tunic adventitia or establishing contact with an outer surface of a vessel. The implantable material of the present invention, when wrapped, deposited or otherwise establishes contact with said outer target zone, that is, the anastomosis or its surroundings, serves to restore homeostasis. That is, the implantable material of the present invention can provide an environment that mimics support physiology and leads to the formation, maturation, integration or stabilization of vascular access structures.

For the purposes of the present invention, contact establishment means interacting directly or indirectly with an extraluminal or non-luminal surface as defined elsewhere in the present specification. In the case of certain preferred embodiments, real physical contact is not required to achieve effectiveness. All that is required to implement the present invention is the extraluminal or non-luminal deposition of an implantable material in, next to, or in the vicinity of an injured or diseased area in an amount effective to treat the injured or diseased area. . In the case of certain diseases or injuries, a diseased or injured area may manifest clinically on an interior surface of light. In the case of other diseases or injuries, an injured or diseased area may manifest on an extraaluminal or non-luminal surface. In some diseases or injuries, a diseased or injured area can manifest clinically both on an interior surface of light and on an extraluminal or non-luminal surface. The present invention is effective for treating any of the above clinical manifestations.

For example, endothelial cells can release a wide variety of agents that, in combination, can inhibit or mitigate adverse physiological events in relation to association with acute complications that follow the creation of a vascular access structure. As exemplified herein, a composition and method of use that recapitulates normal physiology and dosage are useful for improving the formation, maturation, integration or stabilization of a vascular access structure, as well as for promoting persistence to long term of such vascular access structures. Typically, the treatment includes placing the implantable material of the present invention in, next to, or in the vicinity of the area of the vascular access structure, for example, in the perivascular space external to the lumen of the artery and vein involved in the intervention. When wrapped, wrapped around, deposited, or otherwise established contact with an injured, traumatized or diseased blood vessel, the cells of the implantable material can provide growth regulating compounds to the vasculature, for example to the underlying smooth muscle cells within of the blood vessel It is contemplated that, when located in an extraluminal zone, the cells of the implantable material provide a continuous supply of multiple regulatory compounds that can penetrate the vessel tissue and reach the light, and the cells are still protected from the adverse mechanical effects of blood flow in the vessel (or in the vessels). As described herein, a preferred extraluminal zone is an outer surface of a blood vessel.

Treatment with a preferred embodiment of the present invention can stimulate normal or near-normal healing and normal physiology. On the contrary, in the absence of treatment with a preferred embodiment of the present invention, normal physiological scarring is impaired, for example, natural endothelial cells and smooth muscle cells can grow abnormally at an exuberant or uncontrolled rhythm after the creation of a vascular access structure, which leads to adverse clinical consequences, including the failure of the vascular access structure. Accordingly, as contemplated herein, treatment with the implantable material of the present invention will improve healing of natural tissue in the area (or areas) of the anastomosis to maintain the persistence of the vascular access structure.

For the purposes of the present invention, vascular access structures could be shaped in a variety of configurations. Vascular access structures may include arteriovenous fistulas, arteriovenous grafts, peripheral bypass grafts, permanent venous catheters, permanent vascular holes, all of which occur naturally or surgically created, or other vascular anastomosic structures created to improve vascular access in a patient. . Additionally, several embodiments of vascular access structures are formed in a variety of configurations that include laterolateral anastomosis, terminolateral anastomosis, terminoterminal anastomosis, and lateroterminal anastomosis. Vascular access structures can also be placed in a variety of anatomical locations.

The implantable material of the present invention can be placed in a variety of configurations in the vascular access structure to be treated. According to certain embodiments, the implantable material of the present invention can be placed both in the anastomotic junction and also in the proximal vein segment, distal to the anastomosis. In other embodiments, the implantable material of the present invention can be placed in the arterial segment, in the proximal vein segment, or by bridging the vascular access structure. In another embodiment, the implantable material can also be placed in the graft material or in a part of the graft material in the anastomotic junction. The vessels can be fully or partially contacted, for example, the implantable material of the present invention can be applied to the vessels circumferentially or in an arc configuration. A vessel or vascular access structure only needs to be in contact with an amount of implantable material sufficient to improve the formation, maturation, integration or stabilization of the vascular access structure.

Arteriovenous fistula

According to certain embodiments, an arteriovenous fistula (hereinafter "AV fistula") created for vascular access can be treated with the implantable material of the present invention. An AV fistula can be placed in a variety of locations within a patient, including, for example, placement in the neck, wrist, forearm and arm. Clinical configurations of AV fistulas include radiocephalic fistulas (between the radial artery and the cephalic vein), Brescia-Cimino fistulas (a laterolateral anastomosis of the radial artery and the cephalic vein inside the wrist), brachiocephalic fistulas (between the brachial artery and the cephalic vein), brachial-antecubital fistulas (between the brachial artery and the cephalic vein), brachiobasilic fistulas, (a transposed basilic vein), cubitalcephalic fistulas (between the ulnar artery and the cephalic vein) and saphenous loop fistulas (vein) saphenous and right side of the femoral artery).

As a consequence of AV fistula surgery, complications typically occur during three phases. These phases are an acute phase, which is often characterized by a thrombosis, an intermediate phase, whose clinical distinctive is a failure of the fistula in its maturation, and finally a more chronic failure of a functioning fistula, already established, that For example, it may be due to progressive venous stenosis.

The characteristics of AV fistula maturation include, for example, the ability to repeatedly cannulate the fistula for dialysis. Another characteristic of the maturation of an AV fistula is the ability to obtain sufficient blood flow that is useful for hemodialysis. A suitable blood flow is at least an adequate flow rate to support dialysis using a dialysis machine such that no recirculation occurs. A dialysis blood flow sufficient for the purposes of the present invention is a blood flow of at least about 350 ml / minute at a time point no greater than 24 weeks, preferably more than 20 weeks, more preferably 16 weeks, and with the highest preference twelve weeks after the creation of the fistula. Currently, it is understood that the mechanisms of failure of AV fistulas to mature include, for example, early thrombosis of the fistula vessels, stenosis in or near the anastomotic area, the presence of secondary veins, including lateral branches

or collateral veins, inadequate vein size, including an inadequate internal vein diameter, and late fistula failure due to progressive stenosis. Secondary veins can prevent the development of the fistula by diverting the bloodstream and not allowing the vein in association with the fistula to have an adequate size to make cannulation possible. It is currently thought that secondary veins could develop, for example, in response to the presence of stenosis in the fistula. The mechanisms of maturation of AV fistulas are multimodal and generally require the establishment of multiple clinical signs. The absence of stenosis is generally an insufficient indication of a mature AV fistula.

In addition, an AV fistula that is considered adequate for the purpose of dialysis requires both maturation of the tissue, those changes that occur in the vein segment of the fistula that allow the fistula to be repetitively cannulated, such as a sufficient blood flow to endure dialysis An AV fistula may remain persistent even when there is very little blood flow, but a persistent AV fistula may not be clinically suitable for dialysis. For the purposes of the present invention, a clinically suitable blood stream for dialysis is about 150-500 ml / minute, preferably about 300-500 ml / minute, and most preferably about 350-400 ml / minute; suitable blood pressures are about 50-180 mmHG, preferably about 50-120 mmHg.

For the purposes of the present invention, it is believed that treatment with the implantable material of the present invention provides a homeostatic environment sufficient for common complications in maturation of AV fistulas, for example, thrombosis, stenosis, coagulation or secondary vein growth. be reduced when the material is placed next to - or in the vicinity of - the fistula, either at the time of creation of the fistula, or at a later stage. This type of beneficial environment allows an AV fistula to mature or remain in a mature phase. For example, maturation is established functionally when the vein of the AV fistula increases its thickness and is capable of conducting a high blood flow at elevated pressure. The treatment of an AV fistula with the implantable material provided for the present invention improves the maturation of the fistula or prevents the fistula from maturing. It is understood that, for the purposes of the present invention, this improvement in the maturation of the AV fistula includes any improvement in the operation of the fistula, including its formation, the time required to reach a functional state, as well as the maintenance of the fistula. In a mature way.

An immediate post-operative thrombosis can prevent the formation of a persistent AV fistula and lead to premature failure of the fistula. As explained herein, treatment with the implantable material of the present invention at the time of surgery can prevent immediate failure of the fistula due to post-operative thrombosis. For example, the implantable material releases antithrombosis mediators that reduce thrombosis and can maintain a persistent fistula through the formation and maturation phases of the fistula.

Placing implantable material in or near the AV fistula area at the time of surgery can also improve maturation by reducing stenosis at or near the fistula anastomosis, allowing the fistula to reach be of an adequate size to provide sufficient blood flow to support dialysis, facilitate arterial and venous dilation, decrease the formation of parasitic secondary veins, maintain secondary native veins to improve fistula maturation, and improve the size of the come to.

Additionally, an AV fistula requires a longer period of time to reach maturation than an AV graft. During the period of maturation of the fistula, hemodialysis is generally performed using a percutaneous or permanent catheter, which leads to an increased risk of infection and compromises the central permeability of the vein. The placement of the implantable material in the area of an AV fistula at the time of the creation of the fistula can reduce the time required for the maturation of the fistula, thereby reducing the corresponding risks of infection and the compromised central persistence of the vein . The placement of implantable material in the area of a permanent catheter. it can reduce the risk of thrombosis, intimal layer hyperplasia and restenosis in relation to association with the permanent catheter, thereby reducing the risks of infection and compromised central persistence of the vein.

Finally, the use of the implantable material described herein may decrease the late failure of a mature AV fistula. A mature fistula may experience a decrease in blood flow and an increase in venous stenosis due to late progressive stenosis. Stenosis, or occlusion of a fistula to a degree sufficient to reduce blood flow below a level necessary for dialysis, may require interventional angioplasty or a stent of the fistula to restore adequate blood flow levels. Such interventional therapies increase the risk of venous stenosis and occlusion, further preventing the formation of a mature AV fistula. In addition, the stent may result in occlusive thrombosis or in restenosis of the treated vessel in the part of the distal vessel and proximal to the stent, often referred to as edge effects.

The application of the implantable material results in a safe remodeling (a combination of vascular dilation with a simultaneous inhibition of hyperplasia of the venous neointimal layer), thereby preventing late progressive stenosis, increasing the blood flow of the mature fistula, reducing the need of a rehabilitative angioplasty or the stent of an occluded fistula, preventing edge effects in relation to association with the stent, and prolonging the duration and capacity of use of the mature fistula.

The implantable material of the present invention can be provided to the fistula in any of a series of different stages. For example, at the time of surgery you can prevent the AV fistula from maturing or it can improve the ripening of the fistula. The implantable material can also be provided after the initial surgical intervention to accelerate healing in general, as well as after a mature AV fistula has been formed to keep it in a clinically stable state. Additionally, the implantable material can also rescue a mature AV fistula that subsequently fails or may prolong the duration of a mature fistula. These cases are examples without limitation of the improvement of the maturation of an AV fistula. Accordingly, it is contemplated that the implantable material can be used not only at the time of initial surgery to create the AV fistula, but also at subsequent moments (for example, to maintain a mature fistula or rescue a mature fistula so that do not fail). Subsequent administrations can be performed surgically or non-invasively.

Arteriovenous graft. According to further embodiments, an arteriovenous graft (hereinafter "AV graft"), created for vascular access can be treated with implantable material of the present invention. An AV graft may be in the form of a straight forearm graft, a forearm loop graft or a brachial graft. Efferent areas of the artery include, but are not limited to, the common carotid artery, the radial artery in the wrist, the brachial artery in the antecubital fossa, the brachial artery in the lower arm, the brachial artery just below the armpit , axillary artery and femoral artery. Efferent areas of veins include, without limitation, the median antecubital vein, the proximal cephalic vein, the distal cephalic vein, the basilic vein at the level of the elbow, the basilic vein at the level of the upper arm, the axillary vein, the jugular vein and femoral vein. Additional locations, arterial and venous, suitable for AV graft formation, include the chest wall (axillary artery to the subclavian vein), the lower extremities (femoral artery / vein, saphenous vein, or tibial artery (anterior) ), the aorta to the vena cava, the axillary artery to the femoral vein or the femoral artery to the axillary vein.

For the purposes of the present invention, a functional AV graft involving a prosthetic bridge suitable for dialysis is capable of conducting high flow and high pressure blood through the prosthetic bridge. In said AV grafts, the blood flow rate in the venous efferent region of the graft is substantially similar to the blood flow rate downstream of the efferent region of the graft. Blood flow rates suitable for dialysis are approximately 150-500 ml (minute, preferably around 300-500 ml / minute, and most preferably approximately 350,400 ml / minute; adequate blood pressures are approximately 50-180 mmHg, preferably 50-120 mmHg.

AV grafts generally fail due to intimal hyperplasia in association with the graft followed by thrombosis in association with the graft in the vein graft anastomosis or in the proximal venous segment. AV grafts are also vulnerable to failure due to poor tissue integration between the native vessels and the prosthetic bridge material and the eventual dehiscence of the bridge material from the vessels.

For the purposes of the present invention, treatment with the implantable material of the present invention provides a beneficial homeostatic environment such that complications in relation to association with graft integration and maturation, for example, thrombosis, stenosis, coagulation, and / or dehiscence are reduced, either at the time of graft creation, or at a later stage. This type of beneficial environment allows blood vessels in association with an AV graft to fully integrate with the prosthetic bridge material. For example, maturation is functionally established when the AV graft is integrated and is capable of conducting blood with high pressure and a high flow rate. As shown herein, the treatment of an AV graft with implantable material improves graft integration and maturation. For the purposes of this invention, it is understood that the improvement of the integration and maturation of an AV graft includes any improvement in the operation of the graft, including the formation, the time required to reach a functional state, as well as the maintenance of the graft in a functional way

Immediate post-operative thrombosis in association with a graft can prevent tissue integration and eventual formation of a persistent AV graft, and can lead to premature graft failure. As explained herein, treatment at the time of surgery may prevent immediate failure of the AV graft due to postoperative thrombosis. For example, the implantable material releases anti-thrombosis mediators that reduce thrombosis and maintain an unobstructed graft through the graft integration and maturation phases.

The placement of the implantable material in, next to, or in the vicinity of the AV graft anastomosis; in, next to, or in the vicinity of the venous afferent region of the graft; or in the vicinity of the graft at the time of surgery can also improve integration and maturation by reducing immediate thrombosis and progressive stenosis at or near the graft anastomosis. This therapeutic effect allows the graft sufficient time to become adequately integrated with the material of the prosthetic bridge, minimizes thrombosis and occlusion of the blood vessel, and maintains an adequate internal diameter of the vessel to support a sufficient blood flow for dialysis.

The administration of the implantable material can also minimize the late failure of a mature AV graft. A mature graft may experience a decrease in blood flow and an increase in venous stenosis due to late progressive stenosis. The application of the implantable material can result in a safe venous remodeling (a combination of vascular dilation with a simultaneous inhibition of hyperplasia of the venous neointimal layer), thereby preventing late progressive stenosis, increasing the blood flow of the mature graft and prolonging the duration and capacity of use of the mature graft.

As demonstrated herein, the treatment of an AV graft anastomosis with the implantable material of the present invention promotes the formation of a functional AV graft suitable for dialysis. It is further understood that, for the purposes of the present invention, the formation of a functional AV graft includes any improvement in the clinical operation of the graft, or in the process of forming graft anastomosis or in the integration of the prosthetic bridge, or maintenance of graft anastomosis in a mature form, including a reduction in the incidence of dehiscence.

As clinical practitioners fully recognize, the efficacy of an AV graft requires that a graft support and maintain adequate blood flow. In the case of AV grafts useful for dialysis, a suitable blood flow is at least an adequate flow rate to support dialysis using a dialysis machine such that no recirculation occurs. An AV graft that has failed clinically is a graft that cannot support adequate blood flow to support dialysis. It is expected that a preferred embodiment of the present invention will delay the onset of - or decrease the - failure of an AV graft by promoting the formation of a functional AV graft that can withstand a blood flow suitable for dialysis.

Peripheral graft bypass. According to additional embodiments, a peripheral graft created to bypass a failing peripheral blood vessel can be treated with the implantable material of the present invention. A peripheral bypass graft can be placed in a variety of anatomical locations, including limbs such as a region of the leg above or below the knee. A peripheral bypass graft can be used to bypass a blocked peripheral vessel, including a blockage in a peripheral artery or vein. A peripheral bypass graft can be used to restore and / or maintain normal blood flow to the extremities, for example, a sufficient blood flow to maintain normal or near normal peripheral circulation. According to certain embodiments, the peripheral bypass graft is formed from above the block region to below the block region. In certain embodiments, the present invention can be used to improve the functionality, integration, maturation and / or stabilization of a peripheral shunt comprising native materials; In certain others, the graft has a prosthetic bridge.

In the case of a peripheral bypass graft, the implantable material may be placed on an outer surface of the blood vessel at one or both ends of the graft or on an outer surface of the graft material. In certain embodiments, the implantable material may contact the junction of the peripheral bypass graft at one or both ends. In certain other embodiments, the implant may be placed in an outer part of the blood flow downstream of the bypass peripheral graft.

The placement of a preferred embodiment of the implantable material in or near the regions of the inflow or outflow of a peripheral bypass graft at the time of surgery may also improve the formation of a functional graft, promote integration or prevent dehiscence. For the purposes of the present invention, a functional peripheral bypass graft is capable of conducting a normal bloodstream at normal pressures. Normal flow rates for a bypass graft below the knee are approximately 50-150 ml / minute, preferably around 80-100 ml / minute; above the knee, approximately 50-150 ml / minute, preferably around 80-100 ml / minute; for pediatric grafts, approximately 25-30 ml / minute. Suitable blood pressures are approximately 50-180 mmHg, preferably around 50-120 mmHg.

In the case of bypass peripheral grafts treated as described herein, the output flow rate is substantially similar to the input flow rate. The present invention recovers adequate blood flow to the lower extremities and decreases symptoms in association with an inadequate blood flow to the lower extremities. A preferred embodiment of the present invention delays the onset of -o decreases, the failure of a peripheral bypass graft by forming a functional peripheral bypass graft with sufficient blood flow to maintain peripheral circulation.

For the purposes of the present invention, any prosthetic bridge material is suitable for creating a vascular access structure, provided it supports the flow rates and blood pressures required for hemodialysis, in the case of AV grafts, and that supports flow rates and pressures. blood required for peripheral circulation, in the case of peripheral bypass grafts. Typically, prosthetic bridges are preferably flexible and compatible with cell integration, and of the appropriate dimensions to support the required blood flow rates. A preferred embodiment uses a PTFE bridge, or an ePTFE bridge; another uses Dacron® (manufactured by E.I. duPont de Nemours and Co.). Prosthetic bridges can also be constructed of modified PTFE, polyurethane, carbon-coated PTFE, and composite grafts. PTFE grafts can be performed in a variety of physical configurations that include progressively narrowed, stretched, ribbed, smooth, and with multiple levels of PTFE. Prosthetic grafts may also include distal modifications that include venous patches, collars, duckbills, interposed between the artery and the fistula. Additional embodiments may include native materials such as saphenous vein grafts, umbilical vein grafts, femoral vein allografts, and biological xenografts, including grafts for bovine carotid vein and bovine mesenteric vein.

Additionally and in an important way, in the case of an AV graft or a peripheral bypass graft, a normal or almost normal healing rhythm stimulates the endothelial cells to populate the luminal surfaces of the prosthetic bridge, thereby facilitating integration of the graft and vasculature in association with it. To stimulate integration, the therapeutic factors provided by the cells of the implantable material are diffused in the vessel walls. In the case of a synthetic graft material, the porosity of the synthetic material can also affect the ability of the therapeutic factors to reach the cells that proliferate on the luminal surface of a synthetic graft.

PTFE graft. In certain preferred embodiments, a 15-25 cm tube is used. in length and 6 mm inside diameter to form the graft (standard wall PTFE graft Atrium Advanta VS, 0.6 mm, Atrium Medical Corp., Hudson, NH). PTFE, a particularly preferred graft material, is a flexible polymer that has been shown not to be thrombogenic when used in surgical interventions. It is contemplated that alternative polymer materials, such as Dacron®, which has properties similar to those of PTFE, could also be used as graft materials.

The graft could be cut to a desired length to facilitate precise placement in a given patient. The graft could be a forearm loop graft or a straight graft. The graft ends could be cut at an angle, with tabs, or in another configuration, sufficient to increase the surface area of the graft ends to suture or to improve graft accommodation by the determined patient. The graft ends could also be roughened or otherwise modified to facilitate cell adhesion. Additionally, the graft material could be coated with gelatin, albumin, or other therapeutic agent.

Finally, the provision of implantable material to an AV graft or to a peripheral bypass graft that has failed or fails may result in the rehabilitation of the original graft thereby recovering the graft's functionality. In a circumstance related to the case, a native AV fistula that has failed can be replaced with an AV graft in combination with the implantable material of the present invention as an interventional therapy.

General considerations ... In certain embodiments of the invention, additional therapeutic agents were administered before, coinciding with, or after administration of the implantable material. For example, agents that prevent or decrease blood clot formation, thrombocyte aggregation or other similar blockages can be administered. Exemplary agents include, for example, heparan sulfate and the transforming growth factor (hereinafter TGF) �. Other cytokines can also be incorporated into the implantable material.

or growth factors, depending on the clinical indication that the implant needs, including the endothelial vascular growth factor (hereinafter VEGF) to promote re-endothelialization and fibroblast growth factor-b (hereinafter FGF-b) to promote graft integration. Other types of therapeutic agents include, but are not limited to, anti-proliferation agents, and antineoplastic agents. Examples include rapamycin, paclitaxel and the E2F Decoy agent. Any of the above agents can be administered locally or in a generalized manner; if administered locally, certain agents may be contained within the implantable material or contributed by cells.

Additionally, agents with safe tissue remodeling that act as a mediator can be administered in combination with the implementations of implantable material described herein. For example, certain agents may promote a normal or similar to normal light regeneration or a remodeling of the luminal tissue in an area of vascular injury, including surgical areas. Again in this case, said agents may be contained within the implantable material.

or contributed by the cells, by the thickness of the walls of the vessels, maintaining the diameter of light, or a combination of the foregoing, wherein the method comprises the step of locating the implantable material in, next to or in the vicinity of the vascular access structure in an amount effective to meet one or more of the above assessment criteria.

The implantable material of the present invention can be applied to any tubular anatomical structure that requires interventional therapy to maintain homeostasis. Tubular anatomical structures include structures of the vascular system, reproductive system, genitourinary system, gastrointestinal system, pulmonary system, respiratory system and ventricular system of the brain and spinal cord. As contemplated herein, tubular anatomical structures are those that have an inner luminal surface and an extraluminal surface. For the purposes of the present invention, an extraluminal surface may be, without limitation, an outer surface of a tubular structure. In certain structures, the inner luminal surface is a stratum of endothelial cells; In certain other structures, the inner luminal surface is a non-endothelial cell layer.

Source of cells

As described herein, the implantable material of the present invention comprises allograft, xenograft or autogenous cells. In certain embodiments, it can be obtained from living cells from a suitable donor. In other specific embodiments, a source of cells from a corpse or a cell bank can be obtained.

In a presently preferred embodiment, the cells are endothelial cells. In a particularly preferred embodiment, said endothelial cells are obtained from vascular tissue, preferably (without limitation) of arterial tissue. As exemplified below, a type of vascular endothelial cell that is suitable for use is an aortic endothelial cell. Another type of vascular endothelial cell that is suitable for use are endothelial cells of umbilical cord veins. And, another type of vascular endothelial cells that are suitable for use are the endothelial cells of the coronary artery. Still other types of vascular endothelial cells that are suitable for use with the present invention include pulmonary artery endothelial cells and iliac artery endothelial cells.

In another presently preferred embodiment, suitable endothelial cells can be obtained from nonvascular tissue. Non-vascular tissue can be obtained from any tubular anatomical structure as described elsewhere in the present specification, or they can be obtained from any non-vascular tissue or organ.

In yet another embodiment, endothelial cells can be obtained from progenitor cells or stem cells; In yet another embodiment, endothelial cells can be obtained from progenitor cells or stem cells in general. In other preferred embodiments, the cells may be non-endothelial cells that are allograft, xenograft or autogenous obtained from a vascular or non-vascular organ or tissue. The present invention also contemplates any of the above indicated that are genetically altered, modified or manipulated.

In a further embodiment, two or more cell types are cultured together to prepare the current composition. For example, a first cell can be introduced into the biocompatible implantable material and cultured until it is confluent. The first type of cell may include, for example, smooth muscle cells, fibrocytes, stem cells, endothelial progenitor cells, a combination of smooth muscle cells and fibrocytes, and any other type of cells desired or a combination of types of cells that are desired that are suitable to create an environment that leads to endothelial cell development. Once the first type has reached confluence, a second type of cell is seeded on top of the first type of confluent cells in, on or within the biocompatible material and grown until both first and second cell types have Come to the confluence. The second type of cells could include, for example, endothelial cells or any desired type of cells or combination of desired cell types. It is contemplated that the first and second types of cells can be introduced in a staggered manner, or as a single mixture. It is also contemplated that cell density can be modified or the relationship between smooth muscle cells and endothelial cells altered.

To prevent the over-proliferation of smooth muscle cells or other types of cells prone to excessive proliferation, the culture procedure can be modified. For example, following the confluence of the first type of cells, the culture can be coated with a suitable fixation factor for the second type of cells before the introduction of this second type of cell. Exemplary fixation factors include coating the culture with gelatin to improve fixation of endothelial cells. According to another embodiment, heparin can be added to the culture media during the cultivation of the second type of cells to reduce the proliferation of the second type of cells and optimize the predicted relationship between the first type of cells and the second type of cells. For example, after an initial growth of smooth muscle cells, heparin can be administered to control the development of smooth muscle cells in order to achieve a better relationship between endothelial cells and smooth muscle cells.

In a preferred embodiment, a co-culture is created by first seeding an implantable biocompatible material with smooth muscle cells to create vessel structures. Once the smooth muscle cells have reached confluence, endothelial cells are seeded on top of the smooth muscle cells grown in the implantable material to create a simulated blood vessel. This embodiment can be administered, for example, to an AV graft or bypass peripheral graft according to the methods described herein to promote the integration of the prosthetic graft material.

All that is required of the cells of the present invention is that they have one or more preferred functional phenotypes or properties. As described hereinbefore, the present invention is based on the discovery that a cell having an easily identifiable phenotype when in an association relationship with a preferred matrix (which has been described elsewhere in the present memory) may facilitate, restore or otherwise modulate the physiology of vascular endothelial cells or luminal homeostasis in relation to association with the treatment of vascular access structures such as arteriovenous fistulas or arteriovenous grafts.

For the purposes of the present invention, one of said preferred and easily identifiable cell phenotypes of the present invention is an ability to inhibit

or otherwise interfere with the proliferation of vascular smooth muscle cells as measured in the in vitro assays described below. This phenotype will be referred to herein as the "inhibitory phenotype."

Another easily identifiable phenotype presented by the cells of the present composition is that they are anti-thrombosed or that they are capable of inhibiting adhesion and aggregation of thrombocytes. The anti-thrombosis activity can be determined using an in vitro test with heparan sulfate and an in vitro aggregation assay of

thrombocytes described below.

In a typical operational embodiment of the present invention, it is not necessary for the cells to have more than one of the above phenotypes. In certain embodiments, the cells may have more than one of the above phenotypes.

Although each of the aforementioned phenotypes typifies a functional endothelial cell, such as (without limitation) a vascular endothelial cell, a cell that is not endothelial but has such a phenotype (or said phenotypes) is considered similar to an endothelial cell for the purposes of the present invention, and therefore suitable for use with the present invention. Cells that are similar to endothelial cells will also be referred to herein as functional equivalents of endothelial cells; or functional imitations of endothelial cells. Thus, by way of example only, cells suitable for use with the materials and methods described herein also include stem cells or progenitor cells that give rise to endothelial-like cells; cells that are not endothelial in origin and that still functionally behave like an endothelial cell using the parameters specified herein, and cells of any origin that have been genomanipulated or otherwise modified to obtain functionality similar to that of endothelial cells using the parameters specified herein.

Typically, the cells of the present invention have one or more of the aforementioned phenotypes when they are present in confluent, almost confluent or post-confluent populations and in association with a preferred biocompatible matrix such as those described elsewhere herein. As those skilled in the art will appreciate, confluent, almost confluent or post-confluent cell populations are easily identifiable by a variety of techniques, of which the most common and most widely accepted is direct microscopic examination. Other techniques include the evaluation of the number of cells per surface area using standard cell counting techniques such as (without limitation) a hemocytometer or a Coulter counter.

Additionally, for the purposes of the present invention, endothelial-like cells include (without limitation) cells that emulate or mimic functional and phenotypically confluent, almost confluent or post-confluent endothelial cells as measured by the parameters specified in the present memory

Thus, using the detailed description and guidance specified below, the practitioner normally skilled in the art will appreciate how to make, use, test and identify operational embodiments of the implantable material described herein. That is, the teachings provided herein describe all that is necessary to construct and use the implantable materials of the present invention. And furthermore, the teachings provided by the present invention describe all that is necessary to identify, construct and use compositions that contain operationally equivalent cells. Ultimately, all that is required is that compositions containing equivalent cells be effective in treating vascular access structures that respond to the methods described herein. As those skilled in the art will appreciate, equivalent embodiments of the present composition can be identified using only routine experiments together with the teachings provided herein.

In certain preferred embodiments, the endothelial cells used in the implantable material of the present invention are isolated from the aorta of human body donors. Each batch of cells is obtained from a single or multiple donors, extensively tested for endothelial cell purity, biological function, presence of bacteria, fungi, known human pathogens and other accidental agents. The cells are cryopreserved and collected in banks using well known techniques for subsequent expansion in a culture for subsequent formulation in biocompatible implantable materials.

Cell preparation

As indicated above, suitable cells can be obtained from a variety of tissue types and cell types. In certain preferred embodiments, the human aorta endothelial cells used in the implantable material are isolated from the aorta of donor corpses. In other embodiments, porcine cattle aorta endothelial cells (cell applications, San Diego, California, CA) are isolated from the normal porcine cattle aorta by a procedure similar to that used to isolate human aortic endothelial cells. Each batch of cells is obtained from a single or multiple donors, and is extensively tested for endothelial cell viability, purity, biological function, presence of mycoplasma, bacteria, fungi, yeast, known human pathogens and other accidental agents. The cells also expand, characterize and cryopreserve to form a work cell bank in the third to the sixth step using well-known techniques for subsequent expansion in a culture and for subsequent formulation in biocompatible implantable material.

Endothelial cells of human or porcine aortas are prepared in pre-treated T-75 flasks by adding approximately 15 ml per flask of endothelial cell growth medium. Human aorta endothelial cells are prepared in an endothelial cell growth medium (hereinafter EGM-2, Cambrex Biosciences, East Rutherford, N.J.). EGM-2 consists of a basal medium for endothelial cells (hereinafter EBM-2, Cambrex Biosciendes) supplemented with simple aliquots of EGM-2, containing 2% fetal bovine serum (hereinafter FBS). Swine cells are prepared in EBM-2 supplemented with 5% FBS and 50 g / ml gentamicin. The flasks are placed in an incubator maintained at approximately 37 ° C and with a mixture of 5% CO2 / 95% air, and humidity of 90% for a minimum of 30 minutes. One or two vials of the cells are removed from the freezer at -160 ° C -140 ° C and thawed at approximately 37 ° C. Each vial of thawed cells is seeded in two T-75 flasks at a density of approximately 3X103 cells per cm3, preferably, but not less than 1.0 x103 and not more than 7.0x103; and the flasks containing the cells are returned to the incubator. After approximately 6 to 24 hours, the spent medium is removed and replaced by a fresh medium. The medium is changed every two to three days, after that, until the cells reach approximately 85-100% confluence preferably, but not less than 60% and no more than 100%. When the implantable material is intended for clinical application, only an antibiotic-free medium is used in the post-freezing culture of human aorta endothelial cells and in the manufacture of the implantable material of the present invention.

The endothelial cell growth medium is then removed, and the cell monolayer is rinsed with 10 ml of buffered saline hydroxyethyl-piperazine-ethane-sulfonic acid (hereinafter referred to as HEPES). The HEPES is removed, and 2 ml of trypsin is added to separate the cells from the surface of the T-75 flask. Once the separation has occurred, 3 ml of trypsin neutralizing solution (hereinafter TNS) is added to stop the enzymatic reaction. An additional 5 ml of HEPES is added, and the cells are counted using a hemocytometer. The cell suspension is centrifuged and adjusted to a density of, in the case of human cells, approximately 75X106 cells / ml using EGM-2 without antibiotics, or, in the case of porcine cells, approximately 1.50 X 106 cells / ml using EBM-2 supplemented with 5% FBS and 59 �g / ml gentamicin ..

Biocompatible Matrix

In accordance with the present invention, the implantable material comprises a biocompatible matrix. The matrix is permissive for the growth of cells and their fixation to, on or within the matrix. The matrix is flexible and conformable. The matrix may be a solid, semi-solid or flowable porous composition. For the purposes of the present invention, the term "flowable composition" means a composition susceptible to its administration using an injection discharge device or of the injection type such as, without limiting character, a needle, a syringe or a catheter. Other discharge devices that operate by extrusion, ejection or ejection are also contemplated herein. Porous matrices are preferred. A preferred flowable composition is form preservative. The matrix can also be of a flexible flat shape. It can also be in the form of a gel, a foam, a suspension, a particle, a microcarrier, a microcapsule,

or a fibrous structure. A currently preferred matrix is in the form of particles.

The matrix, when implanted on an outer surface of a blood vessel for example, can reside in the implantation zone for at least about 56-84 days, preferably about at least 7 days, more preferably at least about 14 days , and most preferably at least about 28 days before bioerosione.

A preferred matrix is Gelfoam®. (Pfizer, New York, N.Y.), an absorbable jelly sponge (hereinafter "Gelfoam matrix"). The Gelfoam matrix is a porous and flexible surgical sponge prepared from a solution of purified and specially treated pig skin jelly.

According to another embodiment, modifications to the matrix material can be selected to optimize or control the function of the cells, including the phenotype of the cells (eg, the inhibitory phenotype) as described above, when the cells are in association relationship with the matrix. According to one embodiment, modifications to the matrix material include coating the matrix with binding factors or adhesion peptides that increase the ability of cells to inhibit the proliferation of smooth muscle cells, to decrease inflammation, to increase the production of heparan sulfate, to increase the production of prostacyclin, or to increase the production of TGF-1. Exemplary binding factors include, for example, fibronectin, fibrin gel, and covalently fixed cell adhesion ligands (including, for example, arginine-glycine-aspartate (hereinafter RGD) using standard aqueous carbodiimide chemistry. Additional ligands for cell adhesion include peptides having cell adhesion recognition sequences, including (but not limited to) : RGDY, REDVY, GRGDF, GPDSRG, GRGDY and REDV.

According to another embodiment, the matrix is a different matrix from the Gelfoam. Additional exemplary matrix materials include, for example, fibrin gel, alginate, sodium sulfonate and polystyrene microcarriers, collagen coated dextran microcarriers, PLA / PGA copolymers and pHEMA / MMA (with polymer ratios ranging from 1 to 100% for each polymer). According to a preferred embodiment, these additional matrices are modified to include binding factors or peptides for adhesion, as indicated and described above. Exemplary binding factors include, for example, gelatin, collagen, fibronectin, fibrin gel, and ligands for adhesion of covalently fixed cells (including GDPR) using aqueous carbodiimide chemistry. Additional ligands for cell adhesion include peptides that have cell adhesion recognition sequences, including (but not limited to): RGDY, REDVY, GRGDF, GPDSRG, GRGDY and REDV.

According to another embodiment, the biocompatible matrix material is physically modified to improve the fixation of cells to the matrix. According to one embodiment, the matrix is crosslinked to increase its mechanical properties and improve its cell growth and fixation properties. According to a preferred embodiment, an alginate matrix is first crosslinked using calcium sulfate followed by a second crosslinking step using calcium chloride and routine protocols.

According to yet another embodiment, the pore diameter of the biocompatible matrix is modified. A preferred matrix pore diameter is about 25 µm to about 100 µm; preferably about 25 µm to 50 µm; more preferably about 50 µm to 75 µm; even more preferably about 75 cm to 100 cm. Other preferred pore diameters include diameters below about 25 µm and above about 100 µm. According to one embodiment, the pore diameter is modified using a salt leaching technique. Sodium chloride is mixed in a solution of the matrix material and a solvent, the solution is poured into a mold, and the solvent is allowed to evaporate. The matrix and salt block is then immersed in water and the salt is leached leaving a porous structure. The solvent is chosen such that the matrix is in the solution, but not the salt. An exemplary solution includes polylactide (hereinafter PLA) and methylene chloride.

According to an alternative embodiment, carbon dioxide gas bubbles are incorporated into a non-solid form of the matrix and then stabilized with an appropriate surface active agent. Subsequently, the gas bubbles are removed using vacuum, leaving a porous structure.

According to another embodiment, a freeze-drying technique is used to control the diameter of the pores of the matrix, using the freezing rate of the ice particles to form pores of different diameters. For example, a gelatin solution of approximately 0.1-2% swine gelatin

or bovine can be poured into a mold or dish and pre-frozen at a variety of different temperatures and then lyophilized for a period of time. The material can then be crosslinked by using, preferably, ultraviolet light (254 nm)

or by the addition of gluteraldehyde (formaldehyde). Variations in the pre-freezing temperature (for example - 20º C, -80º C or - 180º C) in the freeze drying temperature (dry freezing at approximately - 50º C), and gelatin concentration (0.1% up to 2.0%; the pore diameter is generally inversely proportional to the concentration of gelatin in the solution) can all affect the resulting pore diameter of the matrix material and can be modified to create a preferred material. Those skilled in the art will appreciate that a suitable pore diameter is that which promotes and sustains optimal cell populations that have the phenotypes described elsewhere in the present specification.

Flexible flat shape. As indicated herein, flat forms of biocompatible matrices can be configured in a variety of shapes and sizes, preferably in a shape and size that is adapted for implantation in, next to or in the vicinity of a fistula, graft, peripheral graft, or other vascular access structure and its surroundings and that can conform to the contoured surfaces of the access structures and blood vessels in association with them. According to a preferred embodiment, a single piece of matrix is sized and configured for application to the specific vascular access structure to be treated.

According to one embodiment, the biocompatible matrix is configured in a flexible flat form. An exemplary embodiment configured for administration to a tubular structure such as (but not limited to) a blood vessel or for administration to a vascular access structure such as (but not limited to) to a blood vessel is illustrated in Figure 1. vascular anastomosis In Figure 1 the characteristics of length, width, thickness and surface area have not been drawn to scale or in a proportional manner; Figure 1 is an illustrative non-limiting embodiment.

With reference to Figure 1, a flexible flat shape 20 has been formed from a piece of suitable biocompatible matrix. All that is required is that the flexible flat form 20 be flexible, conformable or adaptable to a contoured outer surface of a tubular structure such as a blood vessel. The flexible flat shape 20 can make contact with an outer surface of a blood vessel, can wrap an outer surface or can be wrapped around an outer surface.

According to an exemplary embodiment illustrated in Figure 2A, the contoured flexible flat shape 20 'can be configured to contain definable regions such as a body 30, connected to a bridge 50, connected to a tongue 40. The tongue 40 is detachable from the body 30 by bridge 50, although the various regions form an adjoining whole. According to another exemplary embodiment, the inner edges of these various regions are intended to define an inner groove 60 made in the contoured flexible flat shape 20 ’. According to a preferred embodiment, these various regions defining the inner groove 60 further define a first termination point 62 within the interior of the contoured flexible flat shape 20 ', a second termination point 64 on an outer edge of the planar shape contoured flexible 20 ', and a width 66. In this particular exemplary embodiment, the first termination point 62 is in a boundary between the tongue 40 and the bridge 50; and the second termination point 64 is in a boundary between the tongue 40 and the body 30.

In certain embodiments, it is contemplated that the width 66 of the groove 60 defined by the tongue 40, body 30 and bridge 50 indicated above is preferably about 0.254 mm (0.01 ") to about 1.016 mm (0.04") , more preferably from about 1.27 mm (0.05 ") to about 2,032 mm (0.08"), and most preferably about 1,524 mm (0.06 "). Preferably, the width 66 of the slot 60 of the flexible flat form 20 'is of a dimension sufficient to discourage the grafted cells from forming an unbroken confluent stratum or a bridge of cells across the width 66 of the slot 60. Without However, it is contemplated that embodiments defining a groove 60 and groove width 66 may be used in the manner described hereinbefore even if the cells span width 66 simply by cutting

or otherwise interrupt said cell layer or said cell bridge.

The present invention further contemplates that the flexible flat shape 20 'of Figure 1 can be used to define a groove 60 immediately before use simply by instructing an experienced practitioner to use a scalpel or other cutting tool to cut the shape flat, partially, defining a groove.

In part, the invention described herein is based on the discovery that a contoured or conformable flexible flat shape allows the implantable material to be optimally applied to a tubular structure without compromising the integrity of the implant or grafted cells in the same. A preferred embodiment optimizes contact with - and conforms to - the anatomy of a surgically treated vessel and controls the extent of the implantable material flap. An excessive flap of implantable material within the space of the adventitious tunic can cause pressure points in the treated vessel, potentially restrict blood flow through the vessel or create other interruptions that could delay or inhibit homeostasis and normal healing. A person skilled in the art will recognize an excessive flap at the time of implantation and will realize the need to reposition or alter, for example by cutting, the implantable material. Additionally, in other embodiments, the flap of the implantable material may result in an overdose of the therapeutic agents dispersed within the implantable material. As described elsewhere in the present specification, chemicals or other therapeutic agents supplied exogenously can be optionally added to an implant. In certain other embodiments, said agents can be added to a biocompatible matrix in the absence of cells; A biocompatible matrix used in this way optionally defines a groove.

In contrast to the above, the implantable material that adequately contacts the target tubular structure may result in insufficient exposure to the clinical benefits provided by the grafted cells or an insufficient dosage of the therapeutic agent added to the implantable material. The person skilled in the art will recognize that a contact less than optimal at the time of implantation needs repositioning or additional implantable material.

Flowable composition

In certain embodiments contemplated herein, the implantable material of the present invention is a flowable composition comprising a biocompatible matrix of particles. Any non-solid flowable composition for use with an injection device of the injectable type capable of either intraluminal (endovascular) administration by navigating the inner length of a blood vessel, or a percutaneous local administration is contemplated herein. . The flowable composition is preferably a composition that retains the shape. Thus, an implantable material comprising cells in, on or within a matrix of particles of the flowable type can be formulated as contemplated herein for use with any injectable discharge device with an inner diameter ranging from about one 22 gauge to about 26 gauge and capable of discharging approximately 50 mg of flowable composition comprising particulate material preferably containing about 1 million cells in about 1 ml to about 3 ml.

According to a presently preferred embodiment, the flowable composition comprises a matrix of biocompatible particles such as Gelfoam® particles, Gelfoam® powder, or powdered Gelfoam® (Pfizer Inc., New York, NY) (hereinafter Hereafter "Gelfoam particles) a product obtained from porcine cutaneous gelatin. According to another embodiment, the particulate matrix is of Cytodex-3 microcarriers (manufactured by Amersham Biosciences, Piscataway, NJ), consisting of denatured collagen coupled to a crosslinked dextran matrix.

According to alternative embodiments, the implantable and biocompatible particle matrix is a modified biocompatible matrix. Modifications include those described above for an implantable matrix material.

Planting of biocompatible matrix cells.

Pre-cut pieces of a suitable biocompatible matrix or an aliquot of a suitable biocompatible flowable matrix are rehydrated by the addition of EGM-2 without antibiotics at approximately 37º C and with 5% CO2 and 95% air for 12 to 24 hours . The implantable material is then removed from its rehydration vessels and placed in individual tissue culture dishes. The biocompatible matrix is seeded at a preferred density of approximately 1.5-2.0 X 105 cells (1.25-1.66 X 105 cells / cm 3 matrix) and placed in an incubator maintained at approximately 37 ° C and with 5% CO2 and 95% air and a humidity of 90% for 3-4 hours to facilitate the fixation of the cells. The seeded matrix is then placed in individual tubular containers (manufactured by American MasterTech, Lodi, GA), each provided with a cap containing a 0.2 µm filter with EGM-2 and incubated at approximately 37 ° C with a 5 % of CO2 and 95% of air. The medium is changed every two or three days, after the above, until the cells have reached the confluence. The cells in a preferred embodiment are preferably 6 steps, but less or more step cells can be used.

Growth curve and confluence of cells. A sample of implantable material is removed on or around days 3 or 4, 6 or 7, 9 or 10, and 12 or 13, the cells are counted and assessed for feasibility, and a curve is constructed and evaluated. growth in order to establish growth characteristics and determine whether confluence, near confluence or post-confluence has been achieved. Figures 3A and 3B show representative growth curves of two of two implantable material preparations comprising implanted batches of porcine aorta endothelial cells. In these examples, the implantable material is in a flexible flat form. In general, a person with normal experience will appreciate the signs of acceptable cell growth at early, medium and late time points, such as observing an increase in the number of cells at the early time points (referring to Figure 3A , between approximately days 2 to 6), followed by an almost confluent phase (referring to Figure 3A, between approximately days 6 to 8), followed by a plateau in cell numbers once the cells have reached confluence (referring to Figure 3A, between approximately days 8 to 10), and the preservation of the number of cells when the cells are post-confluent (referring to Figure 3 A, between approximately days 10 to 14). For the purposes of the present invention, cell populations that are on a plateau for at least 72 hours are preferred.

The cell counts are obtained by a complete digestion of the aliquot of implantable material with a solution of 0.8 mg / ml of collagenase in a solution of trypsin-ethylenediaminetetraacetic acid (hereinafter EDTA). After measuring the volume of the digested implantable material, a known volume of the cell suspension is diluted with 0.4% blue trypan. and the viability by exclusion of the blue trypan is established. Viable, non-viable and total cells are listed using a hematocytometer. Growth curves are constructed by graphically representing the number of viable cells based on the number of days elapsed in the culture. The cells are sent and implanted after reaching the confluence.

For the purposes of the present invention, confluence is defined as the presence of at least about 4 X 105 cells / cm 3 when the implantable material is in a flexible flat form (1.0 X 4.0 X 0.3 cm), and preferably about 7X105 to 1X106 total cells per aliquot (50-70 mg) when in the flexible composition. In both cases, the viability of the cells is at least about 90% preferably, but not less than 80%. If the cells are not confluent by day 12 or 13, the medium is changed, and incubation is continued for another day. This process is continued until the confluence is achieved or up to 14 days after sowing. On day 14, if the cells are not confluent, the batch is discarded. If it is determined that the cells are confluent after making checks during the process, a final change of medium is made. This final change of medium is performed using EGM-2 without red phenol and without antibiotics. Immediately after the change of medium, sterile sealing plugs for transport are placed in the tubes.

Functionality evaluation For the purposes of the invention described herein, the implantable material is further tested for evidence of its functionality before implantation. For example, conditioned media are collected during the cultivation period, to ensure heparan sulfate levels, transformation growth factor �1 (hereinafter TGF-�1), basic fibrocyte growth factor (hereinafter b- FGF), and nitric oxide that are produced by cultured endothelial cells. In certain preferred embodiments, the implantable material may be used for the purposes described herein when the total number of cells is at least about 2, preferably about 4X105 cells / cm3 in a flexible flat form; the percentage of viable cells is at least about 80-90%, preferably � 90%, most preferably at least about 90%; the heparan sulfate in conditioned medium is at least about 0.5-1.0 preferably at least about 1.0 mg / X 106 cells / day. The TGF-�1 in conditioned medium is at least about 200-300, preferably at least about 300 picog / ml / day; b.FGF in conditioned medium is below about 200 picog / ml, preferably no more than about 400 picog / ml.

Heparan sulfate levels can be quantified using a routine spectrophotometric digestion test with dimethythylene bluechondroitinase ABC dye. Total levels of sulfated glycosaminoglycans (hereinafter GAC) are determined using a receptor binding analysis with dimethylmethylene blue (hereinafter ( DMB) in which unknown samples are compared with a standard curve using known amounts of purified chondroitin sulfate diluted in a collection medium.Additional samples of conditioned medium are mixed with ABC chondroitin to digest chondroitin and chondroitin sulfates before DMB color reagent addition All absorbencies were determined at the maximum wavelength absorbency of the DMB dye mixed with the standard GAG, generally around 515-525 nm. The concentration of heparan sulfate per 106 cells per day is calculated. subtracting chondroitin sulfate concentration and of the total concentration of glycosaminoglycan sulfated in conditioned medium samples. The activity of ABC chondroitinase is confirmed by digestion of a sample of purified chondroitin sulfate. Samples of conditioned medium are corrected if less than 100% of purified chondroitin sulfate has been digested. Heparan sulfate levels could also be quantified using an enzyme-linked immunosorbent assay (hereinafter ELISA) using monoclonal antibodies

TGF-�1 and b-FGF levels can be quantified using an ELISA assay that employs monoclonal or polyclonal antibodies, preferably polyclonal. The control collection media can also be quantified using an ELISA assay, and the samples are appropriately corrected for the levels of TGF-�1 and b-FGB present in the control media.

Nitric oxide (NO) levels can be quantified using a standard Griess reaction assay. The transient and volatile nature of nitric acid makes it unsuitable for most detection methods. However, two stable decomposition products of nitric oxide, nitrate (NO3) and nitrite (NO2) can be detected using routine photometric methods. The Griess reaction test enzymatically converts nitrate to nitrite in the presence of nitrate reductase. Nitrite is detected by colorimetry as an azo dye product, which absorbs visible light in the range of approximately 540 nm. The level of nitric oxide present in the system is determined by converting all nitrate into nitrite. in the unknown samples, and then comparing the resulting concentration of nitrite with a standard curve using known amounts of nitrate converted to nitrite.

The preferred inhibitory phenotype indicated above is established using the quantitative tests of heparan sulfate, TGF-�1, NO and b-FGF described above, as well as in vitro quantitative tests of smooth muscle cell growth and thrombosis inhibition indicated then. For the purposes of the present invention, the implantable material is ready for implantation when one or more of these alternative in vitro tests confirm that the implantable material is presenting the preferred inhibitory phenotype.

To assess the inhibition of the growth of smooth muscle cells in vitro, the extent of the inhibition is determined in relation to association with cultured endothelial cells. The porcine or human aorta smooth muscle cells are seeded dispersed in 24-well tissue culture plates in a smooth muscle cell growth medium (SmGM-2, Cambrex BioScience). The cells are allowed to fix for 24 hours. The medium is then replaced with a basal medium for smooth muscle cells (SmBM) containing 0.2% FBS for 48-72 hours until the growth of the cells is stopped. The conditioned media are prepared from post-confluent endothelial cell cultures, diluted in a 1: 1 ratio with 2XSMC growth media and added to the cultures. Each test includes a positive control for the inhibition of smooth muscle cell growth. After three or four days, the number of cells in each sample using a Coulter counter is listed. The effect of conditioned media on the proliferation of smooth muscle cells is determined by comparing the number of smooth muscle cells per well immediately before the addition of conditioned medium with that of three or four days of exposure to the conditioned medium, since control means (standard growth media with and without the addition of growth factors). The magnitude of inhibition in relation to association with samples of conditioned medium is compared with the magnitude of inhibition in relation to association with the positive control. According to a preferred embodiment, the implantable material is considered inhibitor if the conditioned media inhibit approximately 20% of what heparin control is capable of inhibiting.

To evaluate the inhibition of thrombosis in vitro, the level of heparan sulfate is determined in relation to association with cultured endothelial cells. Heparan sulfate has both anti-proliferative and anti-thrombosis properties. Using either the routine dimethylmethyl blue chondroitinase ABC test or an ELISA test (both tests have been described in detail above), the concentration of heparan sulfate per 106 cells is calculated. The implantable material may be used for the purposes described herein when the concentration of heparan sulfate contained in the conditioned medium is at least about 0.5-1.0, preferably at least about 1.0 micrograms / 106 cells / day.

Another method of evaluating thrombosis inhibition involves determining the extent of inhibition of thrombocyte aggregation in vitro in relation to association with thrombocyte-rich plasma. Porcine plasma is obtained by the addition of sodium citrate to swine blood samples at room temperature. The citrated plasma is centrifuged at a gentle rate, to aspirate red (erythrocyte) and white (leukocyte) cells into a pildorite, leaving the thrombocytes suspended in the plasma. The conditioned medium is prepared from post-confluent endothelial cell cultures and added to aliquots of the thrombocyte-rich plasma. A thrombocyte aggregation agent (agonist) is added to the plasma as a control. Thrombocyte agonists commonly include arachidonate, adenosine diphosphate (hereinafter ADP) collagen, epinephrine, and ristocetin. (marketed by Sigma-Aldrich Co., San Luis, MO). An additional aliquot of plasma includes aspirin, heparin, abcisimab (reoPro®, Eli Lily, Indianapolis, IN), tirofiban (Aggrastat®, Merck & Co., Inc., Whitehouse Station, NJ), or eptifibatide (Integrilin®, Millennium Pharmaceuticals , Inc., Cambridge, MA). The resulting thrombocyte aggregation is then measured in all test conditions using an aggregometer. The aggregometer measures thrombocyte aggregation by monitoring optical density. As the thrombocytes are added, more light can pass through the sample. The aggregometer communicates the results in "thrombocyte aggregation units", a function of the rate at which the thrombocytes are added. Aggregation is assessed as maximum aggregation in 6 minutes after agonist addition. The effect of the conditioned medium on the thrombocyte aggregation is determined by comparing the initial aggregation of thrombocytes before the addition of conditioned medium with that after exposure of thrombocyte-rich plasma to the conditioned medium, and to the positive control. The results are expressed as a percentage of the initial values. The magnitude of inhibition in relation to association with samples of conditioned medium is compared with the magnitude of inhibition in relation to association with the positive control. According to a preferred embodiment, the implantable material is considered inhibitor if the conditioned medium inhibits approximately 20% of what the positive control is capable of inhibiting.

When ready for implantation, the implantable material comprising a flexible flat shape is supplied in final product containers, each preferably containing a sterile piece of 1X4X0.3 cm (1.2 cm3) with preferably about 5-8 X105, at least about 4X105 cells / cm3 and at least about 905 viable cells, for example, human aorta endothelial cells obtained from a single donor corpse source, per cubic centimeter in about 45-60 ml, preferably about 50 ml, an endothelial growth medium (for example, an endothelial growth medium (EGM-2) that does not contain phenol red or antibiotics. When porcine aorta endothelial cells are used, the growth medium is also EBM-2 that does not contain red of phenol, but supplemented with 5% FBS and 50 g / ml gentamicin.

In other preferred embodiments, the implantable material comprising a flowable particulate form is supplied in final product containers, including, for example, hermetically sealed tissue culture containers modified with pre-filled filter caps or syringes, each preferably containing approximately 50 -60 mg of grafted particulate material with approximately 7X105 to approximately 1X106 total endothelial cells in about 4560 ml. preferably about 50 ml of endothelial growth medium per aliquot.

Duration in storage of implantable material.

The implantable material comprising a confluent, almost confluent or post-confluent cell population can be maintained at room temperature in a stable and viable condition for at least two weeks. Preferably, said implantable material is maintained in a transport medium of approximately 45-80 ml, more preferably 50 ml, with or without additional FBS. The transport medium comprises EGM-2 without phenol red. FBS can be added to the volume of the transport medium to about 10% FBS, or a total concentration of about 12% FBS. However, since the FBS must be extracted from the implantable material before implantation, it is preferred to limit the amount of FBS used in the transport medium to reduce the duration of rinsing before implantation.

Cryopreservation of implantable material.

The implantable material comprising a confluent population of cells can be cryopreserved for storage and / or transport to the clinic without diminishing its clinical power or integrity after eventual thawing. Preferably, the implantable material is cryopreserved in a 15 ml cryovial (manufactured by Naigene® Naige Nunc INS'1, Rochester, NY) in a solution of approximately 5 ml of CryoStor CS-10 (manufactured by BioLife Solutions, Oswego, NY) containing about 10% dimethyl sulfoxide (hereinafter DMSO), about 2 to 8% Dextran and about 50-75% FBS. The cryovials are placed in a cold water bath in iso-propanol, transferred to a freezer at - 80ºC for 4 hours, and subsequently transferred to a container with liquid nitrogen (-150º C to –165ºC)

The cryopreserved aliquots of the implantable material are then thawed at room temperature for about 15 minutes, followed by an additional approximately 15 minutes in a water bath at room temperature. The material is then washed about three times in a 15 ml wash medium. The washing medium comprises EBM without phenol red and with 50 g / ml gentamicin. The first two rinse procedures are carried out for approximately 5 minutes at room temperature. The final rinse procedure is performed for about 30 minutes at 37 ° C in 5% CO2.

Following the defrosting and rinsing procedures, the cryopreserved material is allowed to stand for about 48 hours in approximately 10 ml of recovery solution. For porcine endothelial cells, the recovery solution is EBM-2 supplemented with 5% FBS and 50 g / ml of 37 ° C gentamicin in 5% CO2. For human endothelial cells, the recovery solution is EGM-2 without antibiotics. Additional post-defrosting conditioning can be performed for at least another 24 hours before use and / or storage or transport packaging.

Immediately before implantation, the medium is decanted and the implantable material is rinsed in a sterile saline solution of approximately 50-500 ml (USP). The medium contained in the final product contains a small amount of FBS to maintain the viability of the cells during transport to a clinical area, if necessary. The FBS has been extensively tested for the presence of bacteria, fungi and other viral agents in accordance with Title 10 of the Federal Code of Regulations (hereinafter CFR): animals and animal products. A rinse procedure is used just before implantation, which decreases the amount of FBS transferred preferably to between 0-80 ng per implant.

The total cell load per human patient will preferably be about 1.6-2.6X104 cells per kg. of body weight, but not less than about 2X103 and not more than about 2X106 cells per kg. of body weight.

As contemplated herein, the implantable material of the present invention comprises cells, preferably vascular endothelial cells that are preferably about 90% viable at a density of preferably about 4X105 cells / cm 3 in a flexible flat form and when they are confluent. , produce a conditioned medium containing heparan sulfate in at least about 0.5-1.0 preferably at least about 1.0 micrograms / 106 cells / day, TGF-�1 in at least about 200-300, preferably at minus about 200 picograms / ml / day, and b-FGF below at least about 210 picograms / ml, preferably no more than about 400 picograms / ml.

Download of implantable material in a flexible flat form.

General consideration. The implantable material can be administered to a vascular access structure in a variety of ways. In accordance with a preferred embodiment, the implantable material is a flexible flat shape cut into a shape and size that suits an implantation alongside a fistula, a graft, a peripheral graft, or other vascular access structure and its surroundings, and which can conform to the contoured surfaces of the access structure and their corresponding blood vessels.

According to a preferred embodiment, an individual piece of implantable material is sized for application to the vascular access structure to be treated. According to another embodiment, more than one piece of implantable material in its flexible flat form, for example two, three, four, five, six, seven, eight or more pieces of matrix material, can be applied to a single location of vascular access Additionally, more than one location along the length of a vascular access structure can be treated with one or more pieces of the implantable material. For example, in the case of an arteriovenous graft, each of the proximal venous anastomosis, distal venous anastomosis and distal venous section can be treated with one

or more pieces of the implantable matrix material.

According to an embodiment without limitation, the implantable material is configured to conform to an outer surface of a blood vessel. An example of a flat non-limiting form is illustrated in Figure 1. With reference to Figure 1, the exemplary flexible flat form 20 has a length 12, a width 14 and a height 16. According to a preferred embodiment, the length 12 of the flexible flat form 20 is about 2 cm to about 6 cm, the width 14 of the flexible flat form 20 is from about 0.1 cm to about 0.5 cm.

According to another embodiment, the flexible flat form 20 can be configured as an anatomically contoured shape that conforms to an outer surface of a blood vessel or a vascular access structure. In Figure 2A, a flat anatomically contoured flexible shape 20 'has been configured configured for administration to a vascular access structure, which is described in more detail below.

As explained elsewhere in this specification, the contoured flexible flat shape 20 'of Figure 2A can be configured in a variety of geometric shapes. For example, according to one embodiment, the contoured flexible flat shape 20 'contains several regions that define a groove 60. According to additional embodiments, the edges of the contoured flexible flat shape and / or the edges of the inner groove form an angle or They are curved. According to another embodiment, the height 16 ’of the contoured flexible flat shape 20’ varies across the length 12 ’or the width 14’. Additionally, there may be one or more of a tongue 40, bridge 50 or slot 60, depending on the configuration and the intended purpose for the contoured flexible flat shape 20 ’. With respect to the characteristic of a groove, a groove can be defined anywhere in, on or within the contoured flexible flat shape 20 ’. A slot can be defined to be uniform in width or vary in width. A slot can be defined as linear, nonlinear, or curved.

With reference to Figures 2B, 2C, 2D and 2E, which represent multiple embodiments of the contoured flexible flat form 20 'of the present invention containing at least one slot 60, the contoured flexible flat form 20' can define one or more of one slot in certain embodiments, and can be used in accordance with the methods described herein. The groove 60 defined on or within a contoured flexible flat shape 20 'can be aligned along any edge of the contoured flexible flat shape 20' or can penetrate inside the contoured shape 20 '. Referring now to Figure 2E, the width 66 or the total shape of the groove 60 in or within the contoured flexible flat shape 20 'can be defined to be of a uniform width or of varying width, and can be defined as linear, nonlinear or curved

With reference to Figures 2F and 2G, the contoured flexible flat shape 20 ’can define a slot 60 or 60’ that has different widths 66 and 66 ’, respectively.

As shown in the Figure, the groove 60 'of Figure 2G and the width 66' are representative of an embodiment in which the practitioner, at the time of implantation, cuts the flexible flat shape 20 'as indicated elsewhere in the present specification, thereby converting it into the flexible flat form 20 'drawn in Figure 2G.

According to one embodiment, a vascular anastomotic terminolateral junction, such as an arteriovenous fistula, can be treated using the implantable material of the invention. The steps of an exemplary method for unloading the implantable material in a flexible flat form to a terminolateral vascular anastomosis have been illustrated in Figures 4A, 4B and 4C.

With reference to Figure 4A, a first piece of implantable material 22 is provided to the vascular access structure by passing one end 34, or a second end 36 of the first piece of implantable material 22 below the anastomosic segment 110 until the center 32 of the first piece of implantable material 22 is in a joint 112 where the vessels 100, 110 are located. Then, the ends 34, 36 are wrapped around a suture line 14 of the joint 112, keeping the implantable material centered on the suture line 114. According to one embodiment, the ends 34, 36 of the first piece of implantable matrix material 22 can overlap each other only enough to hold in place the first piece of implantable material 22 According to another embodiment, the ends 34, 36 of the first piece of implantable matrix material 22 do not overlap each other. The ends 34, 36 of the first piece of implantable matrix material 22 or any other piece of implantable matrix material do not have to meet each other, overlap each other or wrap around the entire circumference of either of the two vessels 100, 110. According to a preferred embodiment, the ends 34, 36 of the first piece of implantable material 22 are wrapped as much as possible around the anastomotic junction 112 without stretching or tearing. All that is required is that adequate coverage of the vessel (or vessels) be achieved. Those skilled in the art will appreciate when the administration of the implantable material has been successfully achieved.

With reference to Figure 4B, according to another embodiment, a second piece of implantable material 24 is optionally applied, with the central part of the second piece of implantable material 24 centered on, or adjacent to, or in the vicinity of the anastomosic joint 112 .. The ends 44, 46 of the second piece of implantable material 24 are wrapped around the vessel 100. As described with respect to the first piece of implantable material 22 in Figure 4A, the ends 44, 46 of the second piece of material of implantable matrix 24 may, but do not need to be, touch, overlap, or wrap around the entire circumference of either of the two vessels 100,110.

With reference to Figure 4C, according to yet another embodiment, a third piece of implantable material 26 is optionally placed in the proximal vessel segment 116 of the treated vessel 100, distal to the anastomotic junction 112. The third piece of implantable material 26, of according to one embodiment, it is placed longitudinally according to the length of the vessel 100 with a first end 54 of the third piece of implantable material 26 at, adjacent to, or in the vicinity of the anastomotic junction 112 and a second end 56 of the third piece of material implantable 26 distal to the anastomotic junction 112. As described with respect to the first piece of implantable material 22 of Figure 4A, the ends 54, 56 of the third piece of implantable matrix material 26 may, but are not required to do so , touch, overlap or wrap around the entire circumference of vessel 100.

In accordance with an alternative embodiment, a single piece of contoured flexible flat form 20 'is provided which defines a groove 60, for example the exemplary contoured shape illustrated in Figure 2A, to a vascular access structure, for example a terminolateral anastomosis. Figure 5 illustrates the implantation of the contoured flexible flat form 20 'of the implantable material defining a groove 60 in, adjacent to, or in the vicinity of a terminolateral anastomosis. When the implantable material is used in an envelope mode, it is contemplated that a single piece of implantable matrix material is suitable for treating both the anastomosis and the adjacent vasculature. Each contoured flexible flat shape 20 'is sized and shaped for application to a particular vascular access structure and, therefore, is pre-formed to provide sufficient coverage and level of endothelial cell factors or therapeutic agent (or agents) ( or therapeutic) to create a homeostatic environment for that particular vascular access structure and its adjacent vasculature.

With reference to Figure 5, in one embodiment, a single contoured shape 20 'defining a groove to an anastomosis is provided by separating the body 30 from the tongue 40. The body 30 is placed along a surface of the vessel main 100. The bridge 50 is placed on a surface of the main vessel 100 and below the branch of the auxiliary vessel 110. Then the tongue 40 is carried around the branched vessel 110 and the tongue 40 is placed along an upper surface of the branched vessel 110.

According to Figure 5, the single flat contoured flexible piece 20 ’contains two reference points 70, 80 (see also Figure 2 A). When administered to the area of a terminolateral anastomosis, as illustrated in Figure 5, the two reference points 70, 80 are aligned. The first reference point 70 is located on the tongue 70 and the second reference point is located on the bridge 50 (see also Figure 2 A). In an embodiment of the contoured flexible flat form 20 'the reference points 70, 80 before implantation are separated by a distance of approximately 12.7 mm (1/2 "), preferably less than about 25.4 mm ( 1 ”), more preferably about 25.4 mm (1”), and most preferably no more than 38.1 mm (1.5 ”). When the contoured flexible flat form 20 ’is administered to the area of an anastomosis, the rotation of the contoured flexible flat form 20’ around the branched vessel 110 allowed by the groove property causes the reference points 70, 80 to align.

According to one embodiment (and referring again to Figures 4A, 4B and 4C), for example, when an arteriovenous graft is treated, the first piece of implantable material 22 and the second piece of implantable material 24 are applied to each one of the proximal and distal venous anastomosis. Additionally, the third piece of implantable material 26 can be placed over the distal vein, upstream of the distal vein anastomosis.

According to yet another exemplary alternative embodiment illustrated in Figure 6, a single piece of implantable material of the flexible flat form 20 is applied to a tubular structure such as a vessel 100. It is contemplated that the implantable material can be applied to such a tubular structure. such as a vessel that does not contain a vascular access structure ... For example, a venous part located downstream of a vascular access structure may experience increased inflammation, thrombosis, restenosis or occlusion of the formation of the vascular access structure or of needle chips in the vascular access structure, downstream of the treated venous part. In such a case, the implantable material of the present invention is capable of treating, managing or improving these conditions that arise at a certain distance from the vascular access structure.

Discharge of implantable material in a flowable composition

The implantable material of the present invention, when it constitutes a flowable composition, comprises a biocompatible matrix of particles and cells, preferably endothelial cells, more preferably vascular endothelial cells, which are approximately 90% viable at a preferred density of approximately 0.8 X 104 cells / mg, more preferably about 1.5 X104 cells / mg, most preferably about 2 X104 cells / mg, and which can produce conditioned media containing heparan sulfate at least about 0.5-1.0 , preferably at least about 1.0 micrograms / 106 cells / day, TGF-�1 at least about 200-300, preferably at least about 300 picograms / ml / day, and b-FGF below about 200 picograms / ml and preferably no more than about 400 picograms / ml; and, present the inhibitor phenotype indicated above.

For the purposes of the present invention, and in general, the administration of the flowable particulate material is located in an area located in, adjacent to or in the vicinity of the vascular access structure. The area of the implantable material deposit is exttraluminal. As contemplated herein, localized extraluminal deposition can be performed as indicated below.

In a particularly preferred embodiment, the flowable composition is administered first percutaneously, entering the perivascular space and then depositing in an extraluminal area using a suitable needle, a catheter or other suitable discharge device of the percutaneous injection type. Alternatively, the flowable composition is administered percutaneously using a needle, catheter or other suitable discharge device in conjunction with an identification step to facilitate discharge to an intended extraluminal zone. The identification stage may occur before or coinciding with the percutaneous discharge. The identification stage can be carried out using intravascular ultrasound, other routine ultrasound, fluoroscopy, or endoscopy methodologies, to name a few. The identification step is optionally carried out and does not require the methods of the present invention to be implemented.

The flowable composition can also be administered intraluminally, that is, endovascularly. For example, the composition can be discharged by any device capable of being inserted into a blood vessel. In this case, said intraluminal discharge device is provided with a device that crosses or penetrates that penetrates the luminal wall of a blood vessel to reach a non-luminal surface of a blood vessel. The flowable composition is then deposited on a non-luminal surface of a blood vessel in, adjacent to, or in the vicinity of the area of the vascular access structure.

It is contemplated herein that a non-luminal surface, also called extraluminal, may include an outer or perivascular surface of a vessel, or it may be within the adventitic tunic, middle tunic or intimate tunic of a blood vessel. For the purposes of this invention, a non-luminal or extraluminal surface is any surface except an interior surface of the light.

The penetrating devices contemplated herein may permit, for example, a single discharge point or a plurality of discharge points arranged in a geometric configuration intended to discharge the flowable composition to a non-luminal surface of a blood vessel without disrupt a vascular access structure. A plurality of discharge points may be arranged, for example, in a circle, in a porthole arrangement,

or in a linear network arrangement, to name just a few. The penetrating device may also be in the form of a stent perforator such as, without limitation, a balloon stent that includes a plurality of discharge points.

According to a preferred embodiment of the invention, the penetrating device is inserted through the inner luminal surface of the blood vessel, either proximal or distal to the area of the vascular access structure. In some clinical cases, the insertion of the penetrating device into the area of the vascular access structure could disturb the vascular access structure or result in the dehiscence of an arteriovenous or peripheral graft. Accordingly, in such cases, care should be taken to insert the penetrating device at a location at a certain distance from the vascular access structure, preferably at a distance determined by the practitioner and considered taking into account the specific circumstances that may arise. hand.

Preferably, the flowable composition is deposited on a perivascular surface of a blood vessel, either in the area of a vascular access structure to be treated, or next to or in the vicinity of the area of a vascular access structure. The composition can be deposited in a variety of locations relative to a vascular access structure, for example, downstream of the anastomosis, on the opposite outer surface of the anastomosis vessel. According to a preferred embodiment, an adjacent area is within about 2 mm to 20 mm of the area of the vascular access structure. In another preferred embodiment, a zone is within about 21 mm to 40 mm; In still another preferred embodiment, a zone is within about 41 mm to 60 mm. In another preferred embodiment, a zone is within about 61 mm to 100 mm. Alternatively, an adjacent area is any adjacent location determined by the practitioner where the deposited composition is capable of presenting an expected effect on a blood vessel in the vicinity of the vascular access structure.

In another embodiment, the flowable composition is deposited directly to an extraluminal area surgically exposed and adjacent to, or in, or in the vicinity of the vascular access structure. In this case, the download is guided and directed by direct observation of the area. Also in this case, the download can be helped by the coincidental use of an identification step described above. Again in this case, the identification stage is optional.

Extraluminal Administration For the purposes of the present invention, the administration of the flowable composition is located in an area adjacent to, in the vicinity of, or in, an area in need of treatment. As contemplated herein, the localized extraluminal deposit can be carried out in the manner indicated below.

The flowable composition is discharged percutaneously using a needle, catheter or other suitable discharge device. Alternatively, the flowable composition is discharged percutaneously coinciding with the use of a guiding method to facilitate discharge to the area in need of treatment. After entering the perivascular space, the physician deposits the flowable composition in an extraluminal zone, adjacent to, or in the vicinity of the area in need of treatment. Percutaneous discharge can be guided and guided optimally by routine ultrasound, fluoroscopy, and endoscopy methodologies, to name just a few.

In another embodiment, the flowable composition is discharged locally, to a surgically exposed extraluminal zone and adjacent to, or in, or in the vicinity of an area in need of treatment. In this case, the discharge is guided and directed by direct observation of the area in need of treatment; also in this case, the download can be helped by the coincidental use of other guiding methods as described above.

Anastomotic sealing agent. In other specific embodiments, the flowable composition of the present invention may additionally serve as an anastomotic sealing agent, specifically, or as a surgical sealing agent in general. In said dual-use embodiment, the composition is also effective for tightly closing the junction of two or more tubular structures or leg tightly closing an empty space in a tubular structure when an outer surface of the structure (or structures) is contacted,

or applied in an arc on an outer surface, or applied circumferentially, This type of sealing agent can eliminate a requirement for sutures that can further damage vascular tissue, for example, and contribute to luminal endothelial trauma. Said sealing agent can also provide additional stability in the vicinity of an anastomosis, thereby reinforcing any suture repair. All that is required is that the properties of the type of sealing agent of this dual-use composition do not interfere with, or impair, the coincident expression of the intended phenotype of the cells and the cell-based functionality of the composition.

For the purposes of certain embodiments with a sealing agent, the flowable composition comprises a biocompatible matrix which in itself comprises a component having properties of a sealing agent, such as, without limitation, a fibrin network, while at the same time it has the properties required to support endothelial cell populations or similar to endothelial ones. Also, the biocompatible matrix per se can have hermetic closing agent properties as well as those required to support a population of cells. In the case of other embodiments, the functionality of the sealing agent can be contributed, at least in part, by the cells. For example, it is contemplated that cells in association with the composition produce a substance that can modify a substrate, such that the substrate acquires properties of a sealing agent, while also presenting or maintaining its cellular functionality. required Certain cells can produce this substance naturally, while other cells can be manipulated to achieve it.

Examples

Example 1: study of human AV fistulas

This example provides experimental examples to test and use a

preferred embodiment of implantable material comprising vascular endothelial cells to improve the maturation of a fistula and / or prevent the failure of a fistula

at maturation Using standard surgical interventions, a .fistula is created

at the expected anatomical location. Then, the implantable material, in a flat form

flexible, is deposited in the perivascular space next to the created fistula

surgically; the details of an exemplary procedure are specified to

continuation. As indicated before, the placement and configuration of the material

Implantable can be varied to suit clinical circumstances. In this

study, at least in Figures 1 or 2A a flexible flat shape has been represented

preferred copy

The experiments and protocols specified below provide sufficient

guided:

1. Evaluate the failure of an arteriovenous fistula at maturation in 3 months. .

For this study, maturation failure is defined as the inability to allow repetitive cannulation of the fistula for dialysis, and to obtain sufficient blood flow for dialysis within the range 35-500 ml / minute, with a preferred blood flow of at least 350 ml / minute, within approximately 12 weeks after the creation of the fistula. Standard clinical techniques will be used.

2.

Evaluate access flow and anatomy (% of the stenosis area) by color flow Doppler ultrasonography on day 5, at 2 weeks, at 1, 3 and 6 months and at subsequent time points.

Decrease in the absolute access flow between the initial measurement (day 5 after surgery) and 6 months after surgery, measured by Doppler ultrasonography with color flow. Magnitude of stenosis determined by Doppler ultrasound at 6 months, when the initial value is compared (day 5 after surgery). , Standard clinical techniques will be used

3.

To evaluate the antibody response of human leukocyte antigens (hereinafter HLA) in relation to association with the use of an allogeneic cell product.

Quantitative immunological determination of the presence of donor HLA antibodies at 5 days, 2 weeks, 1, 3 and 5 months after surgery, compared to pre-surgical levels. Standard clinical techniques will be used.

Specifically, the study includes 10 human uremic patients who have undergone surgery for an arteriovenous fistula. These patients who have undergone surgery for arteriovenous fistula will receive (immediately after surgery) the application of two (2) 1X4X0.3 cm (1.2 cm3) embodiments in a flexible flat form; one (i) placed in the anatomical junction and the other is placed longitudinally on the segment of the proximal vein, distal to the anastomosis. 5 more patients will be enrolled, but they will not receive implants. These 5 patients will be used for comparison with the reference treatment.

Clinical follow-ups will be performed at 5 days, at 2 weeks and at 1, 3 and 6 months. Access flow measurements will be performed using Doppler ultrasonography with color flow on day 5 to establish a baseline level, followed at 2 weeks, 1 month, 3 months, and 6 months after surgery. To patients who present an absolute blood flow less than approximately 350 ml / minute, or have a reduction greater than 25% in blood flow with respect to the previous measurement, or present a stenosis greater than 50% (measured by Doppler ultrasonography ), they will be noted for angiography. A restorative clinical intervention such as an angioplasty for stenosis lesions greater than 50% as determined by angiography will be allowed. Patients with fistulas who are unable to mature within 12 weeks will be noted for radiography. Standard clinical repair interventions, including angioplasty and surgery, will be allowed to repair lateral ramifications or to check the fistula, in order to assist with functional maturation in the fistula that has failed to mature within 12 weeks. The duration of participation in the study for each patient will be 6 months.

Accordingly, a total of 15 patients will be enrolled in this clinical study. 10 patients will each receive 2 implants, and 5 patients receiving reference treatment will be used for comparison. Patients who have an AV fistula for hemodialysis access will also be considered.

The ten patients treated with the implantable material of this invention will each have a standard AV fistula placement, medications, treatments, and implants, according to the following study plan. The first five of these patients will receive two implants in a flexible flat shape, one in the anastomotic area and one placed longitudinally in the segment of the proximal vein, distal to the anastomosis. Following the treatment of the last patient within this first group, an observation period will take place before the treatment of the next group. Following a satisfactory review of the 1 month data of the first five patients, the final 5 patients will be treated.

Five patients will be enrolled in the clinical study and will receive standard AV fistula placement, medications, treatments, but not implantable material. These patients will be used for comparison with a reference treatment and will receive similar immunological and radiographic monitoring as such patients treated with implants.

Conventional AV fistula surgery will be performed in accordance with standard operating techniques. After termination of the fistula, but before implantation, a measurement of the efferent vein diameter will be made.

Forceps without teeth will be used to gently elevate the implantable material flat in the rinse containers. The implantable material will be applied after the access surgery has finished and the blood flow has been established through the fistula, all reference measures having been performed. All bleeding will be controlled and the area to be treated will dry as much as possible before placing the implantable material. The area (or areas) will not be irrigated after implant placement. One or two implants will be used to treat the anastomosis area. The other implant will be used to treat the proximal vein segment, distal to the anastomosis. In certain embodiments, the terminolateral vascular junctions will be treated by passing one end of the implant below the anastomosic segment until the central part of the implant is at the point where the vessels are located. Then, both ends will be wrapped around the suture line, keeping the implant centered over the suture line. The segment of the proximal vein (distal to the venous-arterial anastomosis) will be treated by placing the implantable material longitudinally according to the length of the vein beginning in the anastomotic area. The implantable material will not need to be completely wrapped around the circumference of the vein.

Patients will be followed up with standard nursing procedures during the course of recovery in hospital following AV fistula surgery. Vital signs will be carefully monitored. A record of concomitant medications will be kept. Patients will receive instructions on the requirements for follow-up visits at 5 days, at 2 weeks, and at 1, 3, and 6 months.

Access blood flow will be recorded on day 5 (reference value), at 2 weeks and, thereafter, at 1, 3, and 6 months after surgery. The degree of Doppler ultrasonography stenosis will also be determined on day 5 to establish a reference level, and again at 2 weeks, 1, 3 and 6 months for comparison. A 5 cc whole blood sample will be taken to provide serum for the determination of anti-HLA antibody levels at 5 days, 2 weeks, 1, 3, and 6 months after surgery.

Access blood flow will be determined using Doppler ultrasonography with color flow on day 5 (± 24 hours), to establish a baseline measurement, and at 2 weeks (± 2 days), 1 month (± 4 days), 3 and 6 months (± 7 days) after surgery. To patients who present an absolute blood flow of less than approximately 350 ml / minute, or have a greater than 25% reduction in blood flow with respect to their previous measurement

or have an area of stenosis greater than 50% (measured by Doppler ultrasonography) they will be noted for angiography. A restorative clinical intervention such as an angioplasty for stenosis lesions of a stenosis greater than 50% determined by angiography will be allowed. Patients with fistulas whose maturation has failed after 12 weeks will be noted for x-rays. A standard clinical repair intervention will be allowed including angioplasty and surgery to repair lateral ramifications or to check the fistula, in order to assist in functional maturation in fistulas whose maturation has failed within 12 weeks. This intervention may be followed by the implantation of implantable material to improve the maturation of the revised fistula or to maintain the functionality of the revised fistula and rescue a fistula that fails or has failed.

Expected results of the AV fistula study. It is expected that patients treated with the implantable material of the present invention described above will present one or more indications of an improvement in fistula maturation and / or prevention of fistula failure to mature. Specifically, individually treated patients will have, for example, an improved blood flow, to a sufficient flow rate for dialysis (eg, a blood flow within the range of 35-500 ml / minute and preferably at least 350 ml / minute) or a better ability to repeatedly cancel the fistula for dialysis. Another indication of the maturation of a fistula is the thickness of the walls of the veins; a fistula that is mature or that is maturing satisfactorily has a thickening of the walls of the veins. This will be measured using intravascular ultrasonography (hereinafter IVUS) according to standard clinical techniques. Briefly said, IVUS will be used to measure the thickness of the vein walls and discriminate between the thickness of the intimate or middle layer. The treated or control fistula will be cannulated, and the ultrasound probe will be placed inside the target veins and arteries. . Yet another indication of a fistula that works is an adequate diameter of light. It is expected that the implantable material of the present invention will allow to maintain a suitable diameter of light, thereby allowing a blood flow without impediments in flow rates suitable for effective dialysis, that is, a blood flow that is marginally greater than the pump flow rate. of the dialysis machine, or at least an adequate blood flow to prevent recirculation during dialysis. The diameter of the light will be monitored in series using fistula angiography that begins on day 5 after its creation and after this at least 3 months after the surgical intervention. The narrowing of the lumen after surgery will be correlated with blood flow rates using standard Doppler ultrasonography protocols. It is expected that the implantable material will prevent or delay the narrowing that prevents the circulation of the blood stream below a flow adequate for dialysis as indicated herein. This narrowing of the light that characterizes a fistula that has failed may arise due to stenosis and the corresponding increase in thickness of the intimate layer, or it may arise due to a shrinking or contraction of the vessel without any corresponding increase in thickness. In the case of a real increase in thickness, an angioplasty intervention is currently a standard clinical means; In the case of shrinkage and / or contraction due, for example, to a negative tissue remodeling, dilation is currently a standard clinical intervention. It is expected that a fistula treated with implant does not require angioplasty or dilation.

As a group, treated patients are expected to show at least incremental differences in at least one of the aforementioned signs of maturation compared to controls.

Example 2. Study of AV grafts in animals

This example provides experimental protocols for testing and using a preferred embodiment of the present invention in order to promote the formation of a functional AV graft in animal test patients. Using standard surgical interventions, an AV graft was created between the carotid artery and the jugular vein. Then implantable material was placed in the perivascular space adjacent to each surgically created AV graft anastomosis; the details of an exemplary intervention are specified below. As described above, the placement and configuration of the implantable material can be varied. In this study, the implantable material was in a flexible flat shape, as depicted in Figures 4A, 4B and 4C.

Specifically, the study included 26 trial patients of pigs who underwent surgery for an AV graft. Conventional surgical interventions were performed for AV grafts in accordance with standard operating techniques. The implantable material was applied to the AV graft anastomosis and surroundings, as described below, after the graft surgery was completed and the circulation of the bloodstream through the graft was established.

For each experimental participant who underwent AV graft surgery, a six-mm polytetrafluoroethylene graft (hereinafter PTFE) was placed between the common left carotid artery and the right external jugular vein of the experimental participant An anastomosis was created oblique terminolateral at each end of the graft using a continuous 6-0 polypropylene suture. All experimental participants received intraoperative heparin. and aspirin was administered daily after surgery.

Ten of the experimental participants received implantable material that included aortic endothelial cells on the day of surgery. Five of these implants were applied to each experimental participant. Two of the implants were wrapped around each of the two anastomotic areas. In this circumstance, one end of the implantable material was passed under the anastomotic segment until the central part of the implant was at the point where the vessel and the graft meet. Then both ends were wrapped around the suture line, keeping the implant centered over the suture line. The ends overlapped minimally to hold the material in position. A single additional implant was placed according to the length of the proximal venous segment beginning at the anastomosis of each experimental participant. The implant was not completely wrapped around the circumference of the vein.

The anastomotic areas were wrapped with implantable material, for example, as illustrated in Figures 4A and 4B. Additionally, the proximal venous segment (distal to the venous-arterial anastomosis) was treated by placing the implantable material longitudinally according to the length of the vein beginning in the anastomotic area, for example, as illustrated in Figure 4C.

Ten experimental participants received control implants without cells, wrapped around the anastomotic areas and placed in the proximal venous segment of the graft on the day of surgery; for example, as drawn in Figures 4A, 4B and 4C. 6 additional experimental participants did not receive any type of implant. These 6 experimental patients were used for comparison with the reference treatment. The total cell load based on body weight was approximately 2.5 X105 cells per kg. This cell load is expected to be approximately at least 6 to 10 times the estimated cell load to be used in a human clinical study as described below.

Surgical intervention. A 15 cm longitudinal central neck line incision was made and the common left carotid artery was isolated followed by the right external jugular vein. An 8 cm segment of the vein was released from surrounding tissues and all tributaries at the height of the vein were ligated with 3-0 silk sutures. The left carotid artery was fixed and a circumferential arteriotomy of 7 mm in diameter was performed. An oblique terminolateral anastomosis was performed between the artery and a 6 mm internal diameter PTFE graft using a 6-0 continuous polypropylene suture. Once done, the arterial clamp was removed and the graft was washed under pressure with saline and heparin. Blood flow was introduced through the artery into the graft. The graft was then introduced through a tunnel below the sternocleidomastoid muscles and was carried near the right external jugular vein.

A circumferential venotomy of 7 mm in diameter was performed directly in the external jugular vein. The arteriovenous graft was then completed with an oblique terminolateral anastomosis between the PTFE graft and the right external jugular vein using a 6-0 continuous polypropylene suture (the graft length was between 15 and 25 cm and was recorded at the time of the placement). All tweezers were removed and blood flow was confirmed through the graft. The left carotid artery distal to the PTFE anastomosis was doubly ligated with silk sutures 3

0.

Following the termination of the anastomosis, the PTFE arteriovenous graft was positioned to prevent thread formation. The arteriovenous PTFE graft was cannulated percutaneously with a 22-gauge fin needle just distal to the anastomosis of the carotid artery graft. To confirm the placement, blood was aspirated into the system with a 10 cc syringe. The system was then pressure washed with 10 cc of saline solution. An arc radioscope was then placed on the neck of the animal under study so that the anastomosis of the venous graft and the efferent venous pathway could be visualized. Under continuous radioscopy, 10 to 15 cc of iodinated contrast (Renograffin, full intensity) was injected. Angiography of the radiocinematography was recorded and saved for comparison with the angiogram prior to the animal's slaughter

Once the angiography was finished, the anastomotic areas were wrapped with a 10.16 cm X 10.16 cm (4 ”X 4”) wet gauze sponge. Pressure was maintained on the anastomotic areas for a period of approximately 5 minutes, before removing the gauze sponges and inspecting the anastomotic areas. If the anastomosis had not yet been achieved, as evidenced by the slow bleeding, the area was re-wrapped for another 5 minutes. Additional sutures were placed at the surgeon's discretion if the bleeding in the area was severe. Once the hemostasis was achieved, the neck wound was filled with saline solution and a blood flow analysis was performed with a probe in the distal venous efferent pathway using a 6 mm Transonic flow probe. The saline solution was removed, if necessary, and the anastomoses were dried as much as possible and treated with implantable material comprising aortic endothelial cells and control implants. The areas were not treated with either type of implant until all bleeding had been controlled, the flow through the graft was confirmed, and the area dried as much as possible. Once the intervention was finished, the wound was closed in strata and the animal was allowed to recover from anesthesia.

Heparin was administered before surgery in the form of a 100 U / kg / hour embolate injection plus a continuous infusion of 35 U / kg / hour and maintained until the end of the intervention. Doses were given in additional strokes (100 U / kg) as necessary to maintain the ACT � 200 seconds.

Persistence of grafts.

Graft persistence was confirmed by access flow measurements using color flow Doppler ultrasonography and Transonic flow probe (manufactured by Transonic Systems, Inc., Ithaca, NY) immediately after surgery, 3 to 7 days after surgery, and once a week after the above. The grafts were monitored thoroughly for blood flow.

Pathological procedures.

The experimental animals were anesthetized using sodium pentobarbital (65 mg / kg, intravenous). PTFE grafts were exposed and a digital photograph of the PTFE graft and a venous anastomosis were taken. The PTFE arteriovenous graft was cannulated percutaneously with a 22-gauge fin needle just distal to the anastomosis of the carotid artery graft. To confirm the placement, blood was aspirated into the system with a 10 cc syringe. The system was then pressure washed with 10 cc of saline solution. An arc radioscope was then placed on the neck of the animal, so that the anastomosis of the venous graft and the venous efferent pathway could be visualized. . Under continuous radioscopy, 10 to 15 cc of iodinated contrast (Renograffin, full intensity) was injected. Angiography of the radiocinematography was recorded at angles of 0º and 90º with respect to the PTFE graft. The graft persistence and the degree of stenosis of the venous efferent pathway were determined by a masked reading of the necroscopy angiograms compared to the post-placement angiograms. Angiograms were rated on a scale of 0 to 5 depending on the degree of stenosis observed in them. The classification scheme used was as follows: 0 = 0% stenosis, 1 = 20% stenosis, 2 = 40% stenosis, 3 = 60% stenosis, 4 = 80% stenosis, and 5 = 100% of stenosis. It was anticipated that grafts treated with the implantable material of the present invention would exhibit a decreased stenosis percentage compared to control after examination of the angiograms.

Histology Half of the experimental animals (5 with grafted cell implants, 5 with control implants, 3 without implants) were sacrificed 3 days after surgery. The remaining experimental animals (5 with grafted implants, 5 with control implants, 3 without implants) were sacrificed one month after surgery.

A limited necroscopy was defined in all experimental animals, defined as the macroscopic examination of the administration area, including all anastomosic areas and proximal venous areas, and surrounding tissue including lymph node drainage. Tissue was collected from the main organs, including the brain, lungs, kidneys, liver, heart and spleen, and stored for all experimental animals slaughtered within a month after surgery. The organs were to be analyzed only if unusual findings arose from the macrocopic examination of the external surface of the body, or from the microscopic examination of the administration areas and surrounding tissue. No unusual findings emerged that required further examination of the main organs in any of the animals in the study.

All anastomotic areas of AV graft and surrounding tissues, including 5 cm segments. of each of the anastomosed vein and artery, they were cut, fixed with 10% formalin (or its equivalent) and included in glycolmethyl acrylate (or its equivalent). Using sections of a thickness of approximately 3 mm cut with an arc-shaped stainless steel scalpel (or equivalent), sections of at least three regions were prepared: vein graft anastomosis, artery graft anastomosis, and the efferent venous route. Three sections were made transversely through vein graft anastomosis. Five sections were made through the venous efferent pathway (thus covering 1.5 cm. Of efferent vein). Three sections were made through graft artery anastomosis at 1 mm intervals. These sections were mounted on glass slides (or equivalent) coated with gelatin and stained with hematoxylin and eosin or with Verhoeff elastin dye.

Perivascular and luminal inflammation will be determined both acute (3 day participants) and chronic (1 month participants). Acute inflammation is marked by granulocytes, mainly neutrophils, while chronic inflammation is marked by macrophages and lymphocytes. Additionally, the sections can be stained with the following specific markers: anti-CD45 to identify leukocytes, anti-CD3 to identify T cells, CD79 to identify B cells and MAC387 to identify monocytes or macrophages.

The stained slides will be examined to detect the presence of smooth muscle cells and endothelial cells and to detect indications of integration between arterial or venous anastomosis and artificial graft material. All sections of the isolated tissue, including the graft material, the intimate or pseudo intimate layer, the inner part of the middle layer near the light, the outer part of the middle layer near the adventitia layer, and the adventitia layer for each of the vein graft anastomoses., the artery graft anastomosis, and the venous efferent pathway, will be evaluated and scored. The size of each of the tissue compartments will be measured in microns, for example, the intimate layer, the middle layer and the adventitia layer. Each section will be evaluated to detect the presence or extent of the following criteria. Signs of inflammation will be evaluated, including, but not limited to, the presence and extent of neutrophils, lymphocytes, macrophages, eosinophils, giant cells and plasma cells. The graft sections will be evaluated to detect the presence of fibroblasts, neovascularization, calcification, bleeding, congestion, fibrin, graft fibrosis and graft infiltration. The tissue sections will also be evaluated for signs of degeneration, including, but not limited to, the loss of elastin or the absence of the part

5 of tissue, vacuolation of myofibres of smooth muscles or calcification of tissue. Tissue sections will also be evaluated for the presence of tissue necrosis and foreign matter. Scores will be assigned for each variable on a scale of 0 to 4 (0 = non-significant changes; 1 = minimal changes; 2 = mild changes; 3 = moderate changes; and 4 = intense changes).

10 Additional sections of anastomotic areas of arteriovenous grafts. Only of the animal participants of the 1 month trial will be mounted on glass slides and stained (with Verhoeff elastin) for morphometric analysis. Volume measurements will be taken of the luminal layer, middle layer, intimate layer, and total vessel volume using digital planimetry

15 computerized with a video microscope and customized software for each section. For each section the extent of intimal layer hyperplasia will be determined. One method of quantifying intimal layer hyperplasia is by normalizing the area of the intimate layer by the total area of the vessel wall [intimate layer, in mm2) / (intimate layer + middle layer, in mm2)], or by determining the residual light [

20 (light, in mm2) / (light + intimate layer, in mm2)].

Results for participating animals with AV grafts. Participants treated with the implantable material of the present invention as set forth above presented one or more indications of the formation of a clinically functional AV graft. AV grafts treated in accordance with the materials and methods described herein supported sufficient blood flow rates to allow dialysis. An effective dialysis requires a blood flow that is marginally greater than the pump flow rate of the dialysis machine, or at least an adequate blood flow rate to prevent recirculation during dialysis. Likewise, individually treated participants had a lower incidence of defined dehiscence such as separation of the anastomotic vein or artery from the PTFE graft, and a better integration of the prosthetic bridge defined as a proliferation or migration of smooth muscle cells or endothelial cells in or within the light of the prosthetic bridge. Blood flow outside the AV graft in the venous efferent area was comparable to that in the graft area. As used herein, the term "comparable" means substantially similar for clinical purposes. For example, the expected blood flow is about 150-500 ml / minute, preferably about 300-500 ml / minute, and more preferably about 350-400 ml / minute.

Additionally, the migration of smooth muscle cells or endothelial cells into or within the prosthetic bridge will be measured as an indication of integration. It is contemplated that the implantable material of the present invention will promote the proliferation of smooth muscle cells and the proliferation of endothelial cells, as well as the migration of both into the bridge. Three sections of five micrometers could be obtained through the PTFE graft and stained for SMC actin and evaluated to identify SMC and Factor VIII (von Willebrands Factor) or adhesion molecules of endothelial cells and platelets (hereinafter PECAM) -1 to identify endothelial cells. Endothelial cells will be quantified using microscopy / morphometry and custom software.

Yet another indication of a functioning AV graft is an adequate diameter of light. The implants of the present invention allow the maintenance of a suitable diameter of light by reducing the stenosis of the vessels and thereby allowing a blood flow without obstructions in flow rates suitable for an effective dialysis, that is, an effective dialysis requires a current blood that is marginally greater than the pumping flow of the dialysis machine, or at least an adequate blood flow to prevent recirculation during dialysis. The diameter of the lumen and the percentage of stenosis were monitored using angiography of the arteriovenous graft anastomosis on the day of the creation of the arteriovenous graft and just before sacrifice at 30 days. The narrowing of the lumen after surgery was correlated with blood flow rates using standard Doppler ultrasonography protocols.

The implantable material of the present invention reduced the presence and degree of stenosis of the treated anastomoses compared to the control implants. Table 1, below, presents the percentages of stenosis, determined by angiography, for each participant treated in the study. On average, the implantable material reduced stenosis by ninety-five percent, from 46% in controlled animals to 2.5% in those who received implants ([462.5] / 46 X 100). The results will be confirmed histologically. These studies teach that the present invention prevented or delayed the narrowing that reduces blood flow below an adequate flow rate for dialysis, thereby promoting the functionality of an AV graft anastomosis.

Animal Nº

Group Percentage of stenosis (angle of 0º with the graft) Percentage of stenosis (90º angle with the graft) Average percentage of stenosis

1956 1957 1664 1667 1624 1659 1666 1670

2 2 2 2 3 3 3 3 30% 80% 60% 0% ND 0% 0% 0% 20% 80% 80% 20% 0% 0% 205 0% 25% 80% 70% 10% 0% 0% 10% 0%

Group 2: control implant received only from biocompatible matrix. Group 3: Implantable material received in a flexible flat form comprising cells and biocompatible matrix.

Example 3: clinical study of human AV grafts This example provides experimental protocols for testing and using the invention

10 in order to promote the formation of a functional AV graft in human participants in clinical studies. Using standard surgical interventions, an AV graft anastomosis is created at the intended anatomical location and an ePTFE prosthetic bridge is placed between the arterial and venous anastomoses. The implantable material is then deposited in the perivascular space adjacent to each anastomosis of

15 surgically created graft; the details of an example of surgical intervention are specified below. As discussed above, practitioners skilled in the art may vary the placement or configuration of implantable material in a routine manner. Specifically, the study includes human participants in trials that

20 undergo AV graft surgery. Conventional surgical interventions will be performed in accordance with standard operating techniques. The implantable material of the present invention will be applied to the AV graft anastomoses and their surroundings as described below after the surgical intervention has been completed and the blood flow through the graft has been established.

Human participants in the clinical study will receive one or more parts of the implantable material on the day of the surgical intervention. Two or three of these parts will be applied to each participant. A part of implantable material is wrapped around each anastomotic zone. Then one end is passed under the anastomosic segment until half of the envelope is at the point where the vessel and the graft meet. Then both ends are wrapped around the suture line keeping the implant centered over the suture line. The ends can overlap to hold the material in position. A single additional part of implantable material will be placed in the proximal venous segment of the arteriovenous graft, longitudinally according to the length of the vein starting at the anastomosis, of each participant in the study. The implantable material does not require wrapping completely around the circumference of the vein. The anastomotic areas will be treated with preferred implants, for example, as illustrated in Figures 4A, 4B and 4C, or as illustrated in Figure 5. Additionally, in certain patients, the proximal venous segment (distal to the anastomosis arteriovenous) is treated by placing a preferred implant longitudinally according to the length of the vein beginning in the anastomotic area. It is expected that the total cell load based on body weight will be approximately 2.0 X 104 cells per kg to approximately 6.0 X 104 cells per kg.

Clinical follow-ups will be performed at 5 days, at 2 weeks and at 1, 3 and 6 months. Access blood flow measurements will be required using Doppler ultrasonography with color flow on day 5, to establish a baseline level, followed at 2 weeks, 1 month, 3 months, and 6 months after surgery. To study participants who present an absolute blood flow rate of less than 350 ml / minute, or greater than a 25% reduction in flow rate from the previous measurement, or greater than 50% of the area stenosis (measured by Doppler ultrasonography) will be referred for angiography. A restorative clinical intervention such as angioplasty for stenosis lesions of more than 50% determined by angiography will be allowed.

A graft contrast angiography will be performed, as well as in the arterial and venous anastomotic areas, in the reference line and at 3 months. The diameter of light for each region will be calculated and the maximum value of systolic velocity will be measured.

Expected results of the clinical study of human AV grafts

Participants treated with the implantable material of the present invention as described above are expected to present one or more indications of the formation of a clinically functional AV graft. Specifically, individually treated patients will have, for example, an improved blood flow rate, up to at least a sufficient flow rate for dialysis (for example, a blood flow rate within the range of 35-500 ml / minute and preferably at least 350 ml / minute) , a lower incidence of serous extractions in the peri-graft and pseudoaneurysm, and an improved integration of the prosthetic bridge defined as the proliferation or migration of smooth muscle cells or endothelial cells in or within the light of the prosthetic bridge. The blood flow outside the AV graft in the venous efferent area will be comparable to that in the graft area. The term "comparable" means substantially similar for clinical purposes. For example, the expected blood flow is about 150-500 ml / minute, preferably about 300-500 ml / minute, and more preferably about 350-400 ml / minute.

Additionally, the migration of smooth muscle cells or endothelial cells on or within the prosthetic bridge will be measured by intravascular ultrasonography as an indication of integration. It is expected that the implantable material of the present invention, when used as described herein, will promote a proliferation of smooth muscle cells or a proliferation of endothelial cells, as well as the migration of both into the bridge.

Yet another indication of an AV graft that works is the right diameter of light. It is expected that the implants of the present invention will allow for the maintenance of an adequate diameter of light, thereby allowing a blood flow without obstructions in flow rates suitable for effective dialysis, that is, an effective dialysis requires a blood flow that is marginally greater than the pumping flow of the dialysis machine, or at least an adequate blood flow to prevent recirculation during dialysis. The diameter of the light will be monitored using angiography of the arteriovenous anastomosis at the reference line (approximately 5 days after the creation of the arteriovenous graft) and after this at least 3 months after the surgical intervention. The narrowing of the lumen after surgery will be correlated with blood flow rates using standard Doppler ultrasonography protocols. It is expected that the present invention, when used as described herein, prevents or delays the narrowing that obstructs blood flow below a flow adequate for dialysis as described herein.

In the case of AV grafts, the implantable material of the present invention is expected to prevent or reduce the incidence of dehiscence.

As a group, the treated participants are expected to present at least incremental differences in at least one of these aforementioned indications of functionality, comparing them with the controls.

Example 4: study of peripheral grafts

This example provides experimental protocols for testing and using a preferred embodiment of the present invention in order to promote the formation of a functional peripheral graft in participants in the clinical study. Using standard surgical interventions, a peripheral graft anastomosis is created at the intended anatomical location and a prosthetic ePTFE bridge is placed between the anastomoses. The implantable material is then placed in the perivascular space adjacent to each surgically created peripheral graft anastomosis; the details of an example and surgical intervention are specified below. As discussed above, the placement and configuration of implantable material can be varied.

Specifically, the study includes trial participants who undergo peripheral graft surgery. Conventional surgical interventions will be performed in accordance with standard operating techniques. The implantable material will be applied to the peripheral graft anastomosis and surroundings as described below after completing the surgical procedure and having established the blood flow through the graft.

Participants in the clinical study will receive one or more implantable materials on the day of the surgical intervention. Two or three of these implants will be applied to each participant in the clinical study. One such implant is wrapped around each anastomotic zone. One end of the implantable material is then passed under the anastomotic segment, until the central part of the envelope is at the point where the vessel and the graft meet. The ends are then wrapped around the suture line, keeping the implant centered over the suture line. The ends can overlap each other to hold the material in position. A single additional implant will be placed in the proximal venous segment of the peripheral graft, longitudinally according to the length of the vein starting at the anastomosis, of each participant in the clinical study. The implant does not need to be completely wrapped around the circumference of the vein.

The anastomotic areas will be wrapped with implantable material, for example, as illustrated in Figures 4A, 4B and 4C, or as illustrated in Figure

5. Additionally, the segment of the proximal vessel (distal to the anastomosis) is treated by placing the implantable material longitudinally according to the length of the vessel beginning in the anastomotic area. The total cell load based on body weight will be approximately 2.0 X 104 cells per kg, up to approximately 6.0 X 104 cells per kg.

Clinical follow-ups will be performed at 5 days, at 2 weeks and at 1, 3 and 6 months. Blood flow measurements using Doppler ultrasonography with color flow on day No. 5 will be required to establish a reference, followed at 2 weeks, 1 month, 3 months, and 6 months after surgery. To participants in the clinical study who present an absolute blood flow rate of less than 350 ml / minute, or greater than a 25% reduction in flow rate from the previous measurement, or greater than 50% of stenosis area (measured by ultrasonography Doppler) will be referred for angiography. A restorative clinical intervention such as an angioplasty for stenosis lesions of more than 50% determined by angiography will be allowed.

A graft contrast angiography will be performed, as well as the anastomotic areas. The diameter of light for each region will be calculated and the maximum value of systolic velocity will be measured.

Expected results for participants with peripheral grafts.

Participants treated with the implantable material of the present invention as described above are expected to present one or more indications of clinically functional peripheral graft formation. Peripheral grafts treated in accordance with the materials and methods described herein will support a sufficient blood flow to restore or maintain a clinically acceptable blood circulation. Likewise, individually treated participants will have, for example, a lower incidence of dehiscence defined as the separation of the anastomotic vein from the PTFE graft, or a better integration of the prosthetic bridge defined as proliferation or migration of smooth muscle cells or cells endothelial in or within the light of the prosthetic bridge. The blood flow outside the peripheral graft in the efferent area will be comparable to that in the graft area. As used herein, the term "comparable" means substantially similar for clinical purposes. For example, the expected blood flow is about 150-500 ml / minute, preferably about 300-500 ml / minute, and more preferably about 350-400 ml / minute.

Additionally, the migration of smooth muscle cells or endothelial cells on or within the prosthetic bridge will be measured as an indication of integration. The implantable material of the present invention is expected to promote the proliferation of smooth muscle cells or the proliferation of endothelial cells, as well as the migration of both into the bridge.

Yet another indication of a functioning peripheral graft is an adequate diameter of light. It is expected that the implants of the present invention will allow the maintenance of a suitable light diameter, thereby allowing a blood circulation without obstructions in sufficient flow rates to maintain peripheral circulation. The diameter of the light will be monitored using peripheral graft angiography at the reference line and at least 3 months after graft creation. The narrowing of the lumen after surgery will be correlated with blood flow rates using standard ultrasound protocols with Doppler. It is expected that the implantable material of the present invention will prevent or delay the narrowing that prevents blood flow below an adequate flow rate for peripheral circulation as described herein.

In the case of bypass peripheral grafts, treatment with the implantable material of the present invention is expected to result in blood flow rates that allow clinically acceptable circulation, or approximately normal flow rates. Flows entering and leaving the graft will be comparable. The term "comparable" means substantially similar for clinical purposes. For example, the expected blood flow is about 150-500 ml / minute, preferably about 300-500 ml / minute, and more preferably about 350-400 ml / minute. Additionally, the treatment is expected to promote the proliferation and migration of smooth muscle cells and endothelial cells in the prosthetic or native graft.

In the case of peripheral bypass grafts, the implantable material of the present invention is expected to prevent or reduce the incidence of dehiscence.

As a group, treated patients are expected to present at least

incremental differences in at least one of the indications of functionality

mentioned above compared to controls.

Claims (12)

one.

An implantable material comprising cells and a biocompatible matrix for use in the treatment of an arteriovenous fistula, an arteriovenous graft or a peripheral bypass graft in a patient, comprising the step of locating the implantable material on an outer surface of the fistula or graft, in which the implantable material is effective in promoting a safe remodeling of the fistula or graft, thereby effecting vascular dilation with simultaneous inhibition of hyperplasia of the neointimal venous layer.

2.

The implantable material of claim 1, wherein the cells are endothelial cells or cells that have a phenotype similar to the endothelial

3.

The implantable material of claim 1, wherein the biocompatible matrix is a flexible flat material.

Four.

The implantable material of claim 3, wherein the biocompatible matrix is configured for implantation in an anastomosis.

5.

The implantable material of claim 4, wherein the biocompatible matrix defines a groove or is configured as in Figures 1 or 2A.

6.

The implantable material of claims 1 or 2, wherein the biocompatible matrix is a flowable composition.

7.

The implantable material of claim 6, wherein the biocompatible matrix is a flowable composition that retains the shape.

8.

The implantable material of any one of the preceding claims, wherein the arteriovenous fistula is a native arteriovenous fistula.

9.

The implantable material of any one of the preceding claims, wherein the arteriovenous fistula is radiocephalic, brachiocephalic or brachiobasilic.

10.

The implantable material of any one of the preceding claims, wherein the application of the biocompatible material to the arteriovenous fistula or graft is preceded by - or coincides with - the administration of a therapeutic agent.

eleven.

The implantable material of any one of the preceding claims, wherein the application of the biocompatible material to the arteriovenous fistula or graft is preceded by a physical dilation.

12.

The implantable material of any one of the preceding claims, comprising the step of arranging said implantable material on

an outer surface of said fistula or graft in, adjacent to, or in the vicinity of the venous efferent region thereof.