WO2008057590A2

WO2008057590A2 - Methods and compositions for modulating tissue modeling

Info

Publication number: WO2008057590A2
Application number: PCT/US2007/023566
Authority: WO
Inventors: Helen Marie Nugent; Elazer R. Edelman; Robert M. Tjin Tham Sjin
Original assignee: Pervasis Therapeutics, Inc.
Priority date: 2006-11-07
Filing date: 2007-11-07
Publication date: 2008-05-15
Also published as: WO2008057590A3

Abstract

Disclosed is an implantable material comprising a biocompatible matrix and cells which, when provided to a tubular or non-tubular tissue, structure or organ, can promote functionality and positive tissue modeling generally. For example, implantable material of the present invention can reduce fibrosis, fibroblast proliferation and migration, tissue thickening and/or MMP expression and/or activation. According to the methods disclosed herein, the implantable material is provided adjacent to a surface of a tubular or non-tubular tissue, structure or organ. The materials and methods of the present invention comprise cells, preferably endothelial cells or cells having an endothelial-like phenotype.

Description

METHODS AND COMPOSITIONS FOR MODULATING TISSUE

MODELING

Related Application Data

[0001] This non-provisional patent application claims the benefit under 35 U.S.C. Section 1 19(e) of provisional patent application U.S. S.N. 60/857,458, filed on November 7, 2006; and provisional patent application U. S. S.N. 60/875,626, filed on December 19, 2006; the entire content of each of the foregoing incorporated by reference herein.

Background of the Invention

[0002] Tissues and organs injured during surgical interventions or other disruptive events display a variety of distinct acute and chronic phenomena resulting in negative or constrictive tissue modeling. For example, interventions may result in occlusions, tissue thickening, and extracellular matrix deposition at or adjacent to the site of the intervention. Surgical manipulation such as suturing can also result in direct trauma to the cells of the treated tissue, structure or organ. Injury to the cells of the treated tissue, structure or organ during the intervention can adversely influence healing of the treated structure and result in abnormal or pathological tissue modeling. [0003] The goal of improving outcomes and reducing the incidence of clinical sequelae induced during surgical interventions and other injuries therefore is to locally direct tissue modeling, reduce negative tissue remodeling and/or increase positive or expansive tissue remodeling in the treated tissue. [0004] Studies aimed at decreasing negative tissue remodeling and/or increasing positive tissue remodeling have reached inconsistent conclusions. In fact, at the present time, despite the enormity of this problem, no effective surgical, therapeutic or pharmacologic measures for improving outcomes following surgical intervention are available to clinicians. Clearly a need exists to move ahead in this vital area of patient care.

Summary of the Invention

[0005] The present invention exploits the discovery that an implantable material comprising cells and a biocompatible matrix, when provided locally to a tissue, structure or organ, can reduce or inhibit abnormal or pathological tissue remodeling typified by cell proliferation, migration, fibrosis, abnormal collagen deposition, tissue thickening, and/or MMP expression and/or activation. In accordance with the present invention, the implantable material is located in contact with a surface of a tissue, for example, in contact with a non-luminal or exterior surface of a tubular or non-tubular tissue, structure or organ of the vascular system, adjacent to or in the vicinity of an injury, damage or disease of a tissue, structure or organ. [0006] In one aspect, the invention relates to the use of an implantable material comprising endothelial or endothelial-like cells and a biocompatible matrix for the manufacture of an implant for inhibiting abnormal or pathological tissue remodeling. [0007] According to various embodiments of the invention, the abnormal or pathological tissue remodeling is: inhibited at the site of or in the vicinity of or adjacent to the implant in vivo; MMP-mediated; TIMP-mediated; or mediated by one or more TIMP(s) secreted by the endothelial or endothelial-like cells of the implantable material.

[0008] According to additional embodiments of the invention, the abnormal or pathological tissue remodeling involves all or part of a tubular anatomical structure; the tubular anatomical structure is a vascular structure; the vascular structure is selected from the group consisting of a vein, an artery, an arteriovenous native fistula, an arteriovenous graft or a venous catheter.

[0009] According to further embodiments of the invention, the abnormal or pathological tissue remodeling comprises cellular differentiation, infiltration, migration or proliferation; the cellular differentiation, infiltration, migration or proliferation is MMP-mediated; the abnormal or pathological tissue remodeling comprises extracellular matrix protein degradation; the extracellular matrix protein degradation is MMP-mediated; the abnormal or pathological tissue remodeling comprises fibroblast differentiation, infiltration, migration or proliferation; the fibroblasts are selected from the group consisting of adventitial fibroblasts, medial fibroblasts and myofibroblasts; the abnormal or pathological tissue remodeling comprises fibroblast migration to the intima; the abnormal or pathological tissue remodeling comprises fibrosis; the abnormal or pathological tissue remodeling comprises tissue thickening and/or fibrosis; the abnormal or pathological tissue remodeling causes luminal occlusion or narrowing of all or part of a tubular anatomical structure in vivo; or the inhibition of abnormal or pathological tissue remodeling maintains the patency of all or part of the tubular anatomical structure in vivo.

[0010] According to additional embodiments of the invention, the implant is for adjunctive treatment with: (a) physical dilation of all or part of a tubular anatomical structure; and/or (b) surgical resection of fibrotic tissue; and/or (c) anti-matrix metalloproteinase drug treatment; and/or (d) anti-fibrotic drug treatment; and/or (e) anti-angiogenic drug treatment.

[0011] According to various embodiments, the implant is for the treatment of a patient sub-group in which: inhibition of MMP activity at the implant site is indicated; physical dilation of all or part of a tubular anatomical structure precedes, follows or is coincident with implantation at the site of or in the vicinity of or adjacent to said physical dilation; surgical resection of fibrotic tissue precedes, follows or is coincident with implantation at the site of or in the vicinity of or adjacent to said surgical resection; adjunctive treatment precedes, follows or is coincident with implantation of the implant; adjunctive MMP inhibitor treatment precedes, follows or is coincident with implantation of the implant; or adjunctive antifibrotic drug treatment precedes, follows or is coincident with implantation of the implant. [0012] According to further embodiments, the abnormal or pathological tissue remodeling is inhibited by locating the implant at, adjacent to or in the vicinity of said tissue remodeling in vivo; the implant is introduced at, adjacent to or in the vicinity of a tubular anatomical structure in vivo; the endothelial or endothelial-like cells secrete one or more tissue inhibitor metalloproteinase(s) (TIMP(s)) when implanted in vivo in association with said biocompatible matrix; or the endothelial or endothelial-like cells secrete TIMP-2 when implanted in vivo in association with said biocompatible matrix.

[0013] According to further embodiments, the endothelial or endothelial-like cells are selected from: a confluent population of cells; a near-confluent population of cells; a post-confluent population of cells; and cells which have a phenotype of any one of the foregoing populations of cells; the endothelial or endothelial-like cells are allogenic cells; the biocompatible matrix is a flexible planar material or flowable composition; or the biocompatible matrix is a shape-retaining composition. [0014] In another aspect, the invention relates to the use of an implantable material comprising endothelial or endothelial-like cells and a biocompatible matrix for the manufacture of an implant for treating an individual suffering from or susceptible to a condition which can be improved or prevented by inhibiting tissue remodeling. According to one embodiment, the tissue remodeling is abnormal or pathological tissue remodeling. [0015] In a further aspect, the invention relates to the use of an implantable material comprising endothelial or endothelial-like cells and a biocompatible matrix for the manufacture of an implant for treating an individual suffering from or susceptible to a condition which can be improved or prevented by inhibiting MMP activity. [0016] In another aspect, the invention relates to the use of an implantable material comprising endothelial or endothelial-like cells and a biocompatible matrix for the manufacture of an implant for the treatment or prevention of adventitial fibrosis in a blood vessel. [0017] In a further aspect, the invention relates to a method of inhibiting abnormal tissue modeling, comprising the step of providing to a recipient an implantable material comprising a biocompatible matrix and anchored or embedded endothelial cells, endothelial-like cells, or analogs thereof, wherein said implantable material is provided to said recipient in an amount inhibit abnormal tissue modeling in said recipient.

[0018] In another aspect, the invention relates to an implantable material for inhibiting abnormal tissue modeling. The implantable material comprises a biocompatible matrix and anchored or embedded endothelial cells, endothelial-like cells, or analogs thereof, wherein an effective amount of said implantable material inhibits abnormal tissue modeling in said recipient. [0019] In a further aspect, the invention relates to a method of reducing MMP expression and/or activation, comprising the step of providing to a recipient an implantable material comprising a biocompatible matrix and anchored or embedded endothelial cells, endothelial-like cells, or analogs of any of the foregoing, wherein said implantable material is provided to said recipient in an amount sufficient to reduce MMP expression and/or activation in said recipient. [0020] In another aspect, the invention relates to an implantable material for reducing MMP expression and/or activation, wherein said implantable material comprises a biocompatible matrix and anchored or embedded endothelial cells, endothelial-like cells, or analogs of any of the foregoing, wherein an effective amount of said implantable material reduces MMP expression and/or activation in said recipient. [0021] In a further aspect, the invention relates to a method for the treatment or prevention of fibrosis, the method comprising the step of providing to a recipient an implantable material comprising a biocompatible matrix and anchored or embedded endothelial cells, endothelial-like cells, or analogs of any of the foregoing, wherein said implantable material is provided to said recipient in an amount sufficient to treat or prevent fibrosis in said recipient. According to two embodiments, the fibrosis is fibrosis of a vascular tissue, structure or organ or the fibrosis is adventitial fibrosis of a blood vessel. [0022] In another aspect, the invention relates to a method for the treatment or prevention of luminal narrowing or occlusion of a tubular anatomical structure. The method comprising the step of providing to a recipient an implantable material comprising a biocompatible matri; and anchored or embedded endothelial cells, endothelial-like cells, or analogs of any of the foregoing, wherein said implantable material is provided to said recipient in an amount sufficient to treat or prevent luminal narrowing or occlusion in said recipient. According to one embodiment, the tubular anatomical structure is a structure of the vascular system system.

[0023] In a further aspect, the invention is a composition suitable for use with any of the foregoing methods.

Brief Description of the Drawings

[0024] Figures IA and IB are representative cell growth curves according to an illustrative embodiment of the invention. [0025] Figure 2 is a graphical representation of the expression of matrix metalloproteinase 2 (MMP-2) in stained tissue sections of subjects treated with the implantable material and subjects administered the control material at 3 days and at 1 month. [0026] Figure 3 is a graphical representation of the expression of matrix metalloproteinase 9 (MMP-9) in stained tissue sections of subjects treated with the implantable material and subjects administered the control material at 3 days and at 1 month. [0027] Figure 4 is a Pearson correlation between total MMP-2 expression and lumen diameter of a tubular tissue treated with the implantable material at one month.

[0028] Figure 5 is a graphical representation of the change in venous lumen diameter of subjects treated with the implantable material and subjects administered the control material at 3 days and one month.

Detailed Description of the Invention

[0029] As explained herein, the invention is based on the discovery that a cell- based therapy can be used to treat injured, damaged or diseased tissues, structures or organs. The teachings presented below provide sufficient guidance to make and use the materials and methods of the present invention, and further provide sufficient guidance to identify suitable criteria and subjects for testing, measuring, and monitoring the performance of the materials and methods of the present invention. For purposes of the instant description, a blood vessel is deemed representative, however, the teachings, description, testing criteria, and utilities set forth below are directly applicable to other tubular and non-tubular tissues, structures or organs. [0030] In a blood vessel, luminal loss has been attributed at least in part to smooth muscle cell (SMC) migration from the media into the intima in combination with platelet and leukocyte activation and subsequent extracellular matrix deposition at the lumen side. However, other separate and distinct adverse events, such as fibroblast proliferation and migration, tissue thickening (for example, adventitial thickening), abnormal collagen deposition and fibrosis also contribute to lesion formation, abnormal or pathological tissue remodeling resulting in lumen loss after injury. Upon injury, adjacent fibroblasts proliferate and differentiate into myofibroblasts, which can migrate to the injured area, resulting in tissue thickening. Collagen expression within the fibroblasts/myofibroblasts leads to fibrosis. The foregoing adverse events can directly contribute to abnormal or pathological tissue remodeling. As demonstrated elsewhere herein, the tissue at or adjacent to the site of injury is therefore a therapeutic target for treatment with the implantable material as a modulator of abnormal tissue remodeling and to locally direct positive or normal tissue modeling.

[0031] Placement of the implantable material of the present invention at or adjacent to a surgically treated or otherwise injured tissue, structure or organ is effective at diminishing fibrosis and negative tissue remodeling. In the case of vascular interventions, for example, the implantable material inhibits adventitial fibrosis. The adventitia is a key regulator of vascular remodeling. A wide variety of vessel wall injuries contribute to significant changes in both the adventitia and media, which includes inflammation, apoptosis, cellular proliferation, differentiation and migration into the intima, fibrosis, and expression of matrix metalloproteinase (MMPs). Specifically, adventitial inflammation, abnormal collagen deposition and fibrosis all contribute to lesion formation, negative remodeling and lumen loss after experimental vascular injury. Thus, the adventitia provides a potential therapeutic target after vascular injury.

[0032] Fibroblasts are the main cell type implicated in tissue remodeling. In particular, fibroblasts demonstrate preferential proliferation and migration toward the injury site following injury to the tissue. Increased proliferation of fibroblasts in the surrounding tissue layers and modulation of their phenotype to myofibroblasts, contributes to negative tissue remodeling and, in the case of tubular tissues, structures or organs, constricts the injured tubular tissue, structure or organ and contributes to lumen loss.

[0033] Matrix metalloproteinases (MMPs) are necessary for the migration of cells from the surrounding tissues into the injury site following injury by degrading extracellular matrix proteins. Activated myofibroblasts possess matrix degrading activities, which are regulated by the net balance between MMPs and their endogenous inhibitors, tissue inhibitors of matrix metalloproteinases (TIMPs). TIMPs are able to bind both to activated MMPs and to their inactivated precursors, pro-MMPs. See, Nagase, H., et al. "Matrix Metalloproteinases" J Biol Chem 274(31 ) :21491 -21494 (1999). The upregulation of MMPs and downregulation of TIMPs coincide with negative tissue remodeling following tissue injury. For example, adventitial expression of MMPs increases after vascular injury in AV graft models and facilitates the migration of fibroblasts to the neointima. See, Whatling, C. et al., "Matrix Management: Assigning Different Roles for MMP-2 and MMP-9 in Vascular Remodeling" Arterioscler. Thromb. Vase. Biol. 24: 10-1 1 (2004) and Galis, Z. S. et al., "Matrix Metalloproteinases in Vascular Remodeling and Atherogenesis: The Good, the Bad, and the Ugly" Circ. Res. 90:251-262 (2002). [0034] As demonstrated elsewhere herein, fibrosis also plays an important role in tissue remodeling in tubular and non-tubular tissues, structures or organs. The extent of fibrosis and of MMP expression and activation was reduced in tissues, structures or organs treated with the implantable material of the present invention compared to tissues, structures or organs administered the control material, with a larger and more significant difference seen in MMP-2 expression compared to MMP-9 expression. Increased MMP activity may be necessary for cell migration to occur. Evaluation of the conditioned media of the implantable material demonstrated significant levels of TIMP-2. TIMPs are highly specific for MMPs in general but not for any particular MMP. However, a unique feature of TIMP-2 and TIMP-I is that they bind with high affinity to pro-MMP-2 and pro-MMP-9, respectively.

[0035] In one preferred embodiment, the implantable material of the present invention contains confluent or near confluentendothelial cells that target multiple biologic responses to injury. In contrast, administration of a single therapeutic or chemical agent is, at best, only an effort to respond to a single adverse event. Consequently, when the next symptom or adverse event manifests, another single agent is administered, and so on. Not so with the cell-based material of the present invention. For example, endothelial cells in the implantable material secrete heparan sulfate proteoglycan, TGF-βl and TIMP-2 which are all potent regulatory factors. Virtually all TIMPs form tight 1 : 1 inhibitory complexes with MMPs, and can inhibit extracellular matrix degradation and subsequent fibrosis. In the case of blood vessels, for example, the movement of smooth muscle cells and/orfibroblasts through the media to the injured lumen necessitates degradation of basement membrane and elastic lamina with the aid of MMPs. Additionally, endothelial cells of the implantable material also secrete nitric oxide (NO), a potent regulatory compound. Other studies have demonstrated that NO also decreased MMP activities and increases TIMP secretion in eNOS transfected rat smooth muscle cells. Decreased MMP activity and increased TIMP secretion correlates with inhibition of smooth muscle cell migration. Thus, endothelial cells are able to deliver all endothelial derived compounds, including heparan sulfate, TGF-βi, TIMP-2 and NO, in concert to decrease MMP expression and/or activation and fibrosis and, in the case of tubular tissue, structure or organs, to subsequently increase lumen area via positive remodeling influences. [0036] Accordingly, a cell-based therapy for clinically managing fibroblast proliferation and migration, fibrosis, abnormal collagen deposition, tissue thickening and/or MMP expression and/or activation in tubular and non-tubular tissues, structure or organs, for example, the vascular system, has been developed. An exemplary embodiment of the present invention comprises a biocompatible matrix and cells suitable for use with the treatment paradigms described herein. Specifically, in one preferred embodiment, the implantable material comprises a biocompatible matrix and endothelial cells, endothelial-like cells or functional analogs of endothelial cells. In one embodiment, the implantable material is in a flexible planar form and comprises endothelial cells or endothelial-like cells, preferably human aortic endothelial cells and a biocompatible matrix gelatin sponge, preferably a Gelfoam gelatin sponge (Pfizer, New York, NY, hereinafter "Gelfoam matrix"). According to another preferred embodiment, the implantable material is in a flowable or particulate form and comprises endothelial cells or endothelial-like cells, preferably human aortic endothelial cells and biocompatible matrix gelatin particles or powder, preferably Gelfoam gelatin particles or powder (Pfizer, New York, NY, hereinafter "Gelfoam particles").

[0037] Implantable material of the present invention comprises cells engrafted on, in and/or within a biocompatible matrix. Engrafted means securedly attached via cell to cell and/or cell to matrix interactions such that the cells withstand the rigors of the preparatory manipulations disclosed herein. As explained elsewhere herein, an operative embodiment of implantable material comprises a near- confluent, confluent or post-confluent cell population having a preferred phenotype. It is understood that embodiments of implantable material likely shed cells during preparatory manipulations and/or that certain cells are not as securedly attached as are other cells. All that is required is that implantable material comprise cells that meet the functional or phenotypical criteria set forth herein. [0038] The implantable material of the present invention was developed on the principals of tissue engineering and represents a novel approach to addressing the above-described clinical needs. The implantable material of the present invention is unique in that the viable cells engrafted on, in and/or within the biocompatible matrix are able to supply to the treated tubular or non-tubular tissue, structure or organ multiple cell-based products in physiological proportions under physiological feed-back control. As described elsewhere herein, the cells suitable for use with the implantable material are endothelial cells, endothelial-like cells or functional analogs of endothelial cells. Local delivery of multiple compounds by these cells and physiologically-dynamic dosing provide more effective regulation of the processes responsible for maintaining a functional tissue, structure or organ and diminishing injured tissue, structure or organ complications and/or failure. [0039] Importantly, in the case of administration to a vascular structure such as a blood vessel, the endothelial cells, for example, of the implantable material of the present invention are protected from the erosive blood flow within the blood vessel lumen because of its placement at a non-luminal surface of the vessel, for example, at the adventitia or contacting an exterior surface of a vessel. The implantable material of the present invention, when wrapped, deposited or otherwise contacted with such an exterior target site serves to reestablish homeostasis. That is, the implantable material of the present invention can provide an environment which mimics supportive physiology and is conducive to promoting and maintaining optimal tissue modeling events. The implantable material promotes normal tissue modeling and prevents, interrupts or mitigates abnormal or pathological tissue modeling.

[0040] For purposes of the present invention, contacting means directly or indirectly interacting with a tissue surface as defined elsewhere herein. In the case of certain preferred embodiments, actual physical contact is not required for effectiveness. In other embodiments, actual physical contact is preferred. All that is required to practice the present invention is deposition of an implantable material at, adjacent to or in the vicinity of an injured or diseased tissue site in an amount effective to treat the injured or diseased tissue site. In the case of certain diseases or injuries, a diseased or injured tissue site can clinically manifest on an interior or luminal surface. In the case of other diseases or injuries, a diseased or injured site can clinically manifest on an exterior or non-luminal surface. In some diseases or injuries, a diseased or injured site can clinically manifest on both an interior or luminal surface and an exterior or non-luminal surface. The present invention is effective to treat any of the foregoing clinical manifestations. The implantable material of the present invention can be administered to tubular or non-tubular tissues, structures or organs prior to, coincident with, or following diagnosis of a clinical or pathological condition or disease state or a surgical intervention. [0041] For example, endothelial cells can release a wide variety of agents that in combination can inhibit or mitigate adverse physiological events associated with acute complications following tissue injury or surgical intervention. As exemplified herein, a composition and method of use that recapitulates normal physiology and dosing is useful to enhance injured tissue stabilization, as well as promote long-term patency of the injured tissue, structure or organ. Typically, treatment includes placing the implantable material of the present invention at, adjacent to or in the vicinity of the tubular or non-tubular tissue, structure or organ site, for example, in the case of an AV graft anastomosis, in the perivascular space external to the lumen of the artery and vein involved in the procedure. When wrapped, wrapped around, deposited, or otherwise contacting a surface of an injured, traumatized or diseased tissue, the cells of the implantable material can provide growth regulatory compounds to the tissue. It is contemplated that, when situated at an adjacent or external site, the cells of the implantable material provide a continuous supply of multiple regulatory compounds which can penetrate adjacent or surrounding tissue and reach the injured tissue. Yet, in the case of a blood vessel, the cells are protected from the adverse mechanical effects of blood flow in the vessel. [0042] Treatment with a preferred embodiment of the present invention can encourage 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 healing is impaired, e.g., native fibroblasts can grow abnormally at an exuberant or uncontrolled rate following injury, damage, disease or surgical intervention of a tubular or non-tubular tissue, structure or organ, leading to adverse clinical consequences, including tubular or non-tubular tissue, structure or organ failure due to negative remodeling. Accordingly, as contemplated herein, treatment with the implantable material of the present invention will improve the healing of native tissue at the treated site to maintain tubular or non-tubular tissue, structure or organ patency. [0043] The implantable material of the present invention can be placed in a variety of configurations at the tubular or non-tubular tissue, structure or organ to be treated. According to certain embodiments, the implantable material of the present invention can be placed both at the portion of the tubular or non-tubular tissue, structure or organ to be treated or proximal or distal to the portion of the tissue, structure or organ to be treated. The tubular or non-tubular tissues, structures or organs can be contacted in whole or in part, for example, the implantable material of the present invention can be applied to the tissues, structures or organs circumferentially or in an arc configuration. A tubular or non-tubular tissue, structure or organ need only be in contact with an amount of implantable material sufficient to improve the condition of the tissue, structure or organ. [0044] For purposes of the present invention, it is believed that treatment with the implantable material of the present invention provides a beneficial homeostatic environment such that complications common in interventions associated with tubular and non-tubular tissues, structures or organs, for example, tissue thickening, lumen loss, clotting and/or tissue or organ failure are reduced when placed adjacent to or in the vicinity of the tubular or non-tubular tissue, structure or organ whether at the time of the intervention or at a later stage.

[0045] In the case of treated blood vessels, application of the implantable material can result in positive tissue remodeling thereby preventing late progressive lumen loss, increasing blood flow of the mature treated vascular tissue, structure or organ, reducing the need for rehabilitative angioplasty or stenting of an occluded vascular tissue, structure or organ, and prolonging the lifetime and usability of the treated vascular tissue, structure or organ. [0046] The implantable material of the present invention can be provided to the treated tubular or non-tubular tissue, structure or organ at any of a number of distinct stages. For example, treatment at the time of surgery can prevent the tubular or non- tubular tissue, structure or organ from failing to heal and/or can enhance healing of the treated tissue, structure or organ. The implantable material can also be provided after the initial surgery to hasten healing generally, as well as to maintain the tissue, structure or organ in a clinically stable state. Additionally, the implantable material can also rescue a treated tubular or non-tubular tissue, structure or organ that subsequently fails and/or can extend the lifetime of a treated tubular or non-tubular tissue, structure or organ. These situations are non-limiting examples of treatment with the implantable material of the present invention. Accordingly, it is contemplated that the implantable material can be used not only prophylactically but also at the time of initial surgical intervention as an adjunctive therapy, and also at subsequent time points as an interventional therapy (e.g., for maintaining a tubular or non-tubular tissue, structure or organ following intervention or rescuing a treated tubular or non-tubular tissue, structure or organ from failing). Subsequent administrations can be accomplished surgically or non-invasively. [0047] The term "indicated" is a term of art used herein in relation to a patient sub-group to convey the clinical desirability or necessity of a particular intervention in relation to that patient sub-group or population. Thus, references herein to a patient sub-group "in which the inhibition of matrix metalloproteinase (MMP) activity at the implant site is indicated" is intended to define a collection of individuals (e.g. human individuals) in which inhibition of MMP activity is either clinically desirable or necessary. This is the case, for example, where inhibition of MMP activity is palliative, preventative or (at least partially) curative of a disease or condition.

[0048] As used herein, the term "inhibition", as applied to MMP activity, is intended to define a change in the level of biological activity of the MMP enzyme(s). Thus, modulation encompasses physiological changes which affect a decrease in MMP activity. The inhibition may arise directly or indirectly, and may be mediated by any mechanism and at any physiological level, including for example at the level of gene expression (including for example transcription, translation and/or post-translational modification), at the level of expression of genes encoding regulatory elements which act directly or indirectly on the levels of MMP activity, or at the level of enzyme activity (for example by allosteric mechanisms, competitive inhibition, active-site inactivation, perturbation of feedback inhibitory pathways, etc.). Thus, MMP inhibition may imply suppressed expression or under- expression of the gene(s) encoding one or more MMP(s), and/or decreased expression at the transcriptional level. The terms "inhibited" and "inhibit" in relation to MMP activity are to be interpreted accordingly.

[0049] As used herein, the term "mediated", as used in relation to MMPs or TIMPs in the context of any physiological process (e.g. tissue remodeling), disease, state, condition, therapy or treatment is intended to operate limitatively so that the various processes, diseases, states, conditions, therapies or treatments are those in which the MMPs or TIMPs play a biological role. The biological role played by the MMP or TIMP may be direct or indirect and may be necessary and/or sufficient for the manifestation of the symptoms of a disease, state or condition (or its etiology or progression). [0050] As used herein, the term "abnormal", as applied to tissue remodeling, is intended to define tissue remodeling processes which do not occur in healthy individuals. Such processes may therefore be associated with disease or at least some degree of morbidity. As used herein, the term "pathological", as applied to tissue remodeling, is intended to define tissue remodeling processes which give rise to at least some degree of morbidity. Such processes may comprise normal responses to injury, surgical intervention or other forms of medical intervention

(such as drug treatment). Thus, not all pathological tissue remodeling processes are necessarily "abnormal", as herein defined. [0051] General Considerations. In certain embodiments of the invention, additional therapeutic agents are administered prior to, coincident with and/or following administration of the implantable material. For example, agents which prevent or diminish blood clot formation, platelet aggregation or other similar blockages can be administered. Exemplary agents include, for example, heparan sulfate and TGF-β. Other cytokines or growth factors can also be incorporated into the implantable material, depending on the clinical indication necessitating the implant, including VEGF to promote reendothelialization and b-FGF to promote graft integration. Other types of therapeutic agents include, but are not limited to, antiproliferative agents and antineoplastic agents. Examples include rapamycin, paclitaxel and E2F Decoy agent. Additional types of therapeutic agents include TIMPs or synthetic broad band MMP inhibitors. Examples include BB2893 and Marimastat (British Biotech Pharmaceuticals Limited, Oxford, UK), Bay 12-9566 (Bayer, West Haven, CT), AG3340 (Agouron, LaJoIIa, CA), CGS27023A (Novartis, East Hanover, NJ), and COL-3 (Collagenex Pharmaceuticals, Newtown, PA). Any of the foregoing can be administered locally or systemically; if locally, certain agents can be contained within the implantable material or contributed by the cells. [0052] Additionally, agents which mediate positive tissue remodeling can also be administered in combination with the implantable material embodiments described herein. For example, certain agents can promote normal or normal-like tubular or non-tubular tissue, structure or organ regeneration or remodeling of tissue at a site of injury, including surgical sites. Again, such agents can be contained within the implantable material or contributed by the cells. [0053] Accordingly, the present invention also provides for methods of accomplishing surgical and other intervention-related clinical endpoints including improving tissue, structure or organ patency, promoting normal tissue wall thickness, maintaining or increasing lumen diameter, and/or a combination of the foregoing, wherein the method comprises the step of locating the implantable material at, adjacent to or in the vicinity of the tubular or non-tubular tissue, structure or organ in an amount effective to accomplish one or more of the foregoing endpoints. [0054] The implantable material of the present invention can be applied to any tubular or non-tubular tissue, structure or organ requiring interventional therapy to maintain homeostasis. As contemplated herein, tubular anatomical structures are those having an interior luminal surface and an extraluminal surface. For purposes of the present invention, an extraluminal surface can be but is not limited to an exterior surface of a tubular structure. In certain structures, the interior luminal surface is an endothelial cell layer; in certain other structures, the interior luminal surface is a non-endothelial cell layer. Non-tubular anatomical structures include solid organs and hollow organs

[0055] Cell Source. As described herein, the implantable material of the present invention comprises cells. Cells can be allogeneic, xenogeneic or autologous. In certain embodiments, a source of living cells can be derived from a suitable donor. In certain other embodiments, a source of cells can be derived from a cadaver or from a cell bank. [0056] In one currently preferred embodiment, cells are endothelial cells. In a particularly preferred embodiment, such endothelial cells are obtained from vascular tissue, preferably but not limited to arterial tissue. As exemplified below, one type of vascular endothelial cell suitable for use is an aortic endothelial cell. Another type of vascular endothelial cell suitable for use is umbilical cord vein endothelial cells. And, another type of vascular endothelial cell suitable for use is coronary artery endothelial cells. Yet other types of vascular endothelial cells suitable for use with the present invention include pulmonary artery endothelial cells and iliac artery endothelial cells. [0057] In another currently preferred embodiment, suitable endothelial cells can be obtained from non-vascular tissue. Non-vascular tissue can be derived from any tubular anatomical tissue, structure or organ as described elsewhere herein or can be derived from any non-vascular tissue, structure or organ. [0058] In yet another embodiment, endothelial cells can be derived from endothelial progenitor cells or stem cells; in still another embodiment, endothelial cells can be derived from progenitor cells or stem cells generally. In other preferred embodiments, cells can be non-endothelial cells that are allogeneic, xenogeneic or autologous derived from vascular or non-vascular tissues, structures or organs. Exemplary non-endothelial cells include, but are not limited to, epithelial cells. The present invention also contemplates any of the foregoing which are genetically altered, modified or engineered.

[0059] In a further embodiment, two or more types of cells are co-cultured to prepare the present composition. For example, a first cell can be introduced into the biocompatible implantable material and cultured until confluent. The first cell type can include, for example, smooth muscle cells, fibroblasts, stem cells, endothelial progenitor cells, a combination of smooth muscle cells and fibroblasts, any other desired cell type or a combination of desired cell types suitable to create an environment conducive to endothelial cell growth. Once the first cell type has reached confluence, a second cell type is seeded on top of the first confluent cell type in, on or within the biocompatible matrix and cultured until both the first cell type and second cell type have reached confluence. The second cell type may include, for example, endothelial cells or any other desired cell type or combination of cell types. It is contemplated that the first and second cell types can be introduced step wise, or as a single mixture. It is also contemplated that cell density can be modified to alter the ratio of smooth muscle cells to endothelial cells. [0060] To prevent over-proliferation of fibroblasts, smooth muscle cells or another cell type prone to excessive proliferation, the culture procedure can be modified. For example, following confluence of the first cell type, the culture can be coated with an attachment factor suitable for the second cell type prior to introduction of the second cell type. Exemplary attachment factors include coating the culture with gelatin to improve attachment of endothelial cells. According to another embodiment, heparin can be added to the culture media during culture of the second cell type to reduce the proliferation of the first cell type and to optimize the desired first cell type to second cell type ratio. For example, after an initial growth of smooth muscle cells, heparin can be administered to control smooth muscle cell growth to achieve a greater ratio of endothelial cells to smooth muscle cells. [0061] All that is required of the cells of the present composition is that they exhibit one or more preferred phenotypes or functional properties. As described earlier herein, the present invention is based on the discovery that a cell having a readily identifiable phenotype when associated with a preferred matrix (described elsewhere herein) can facilitate, restore and/or otherwise modulate cell physiology and/or tubular or non-tubular tissue, structure or organ homeostasis associated with treatment of tubular and non-tubular tissues, structures or organs. [0062] For purposes of the present invention, one such preferred, readily identifiable phenotype typical of cells of the present invention is an ability to inhibit or otherwise interfere with vascular smooth muscle cell proliferation as measured by the in vitro assays described below. This is referred to herein as the inhibitory phenotype.

[0063] Another readily identifiable phenotype exhibited by cells of the present composition is that they are anti-thrombotic or are able to inhibit platelet adhesion and aggregation. Anti-thrombotic activity can be determined using an in vitro heparan sulfate assay and/or an in vitro platelet aggregation assay described below. [0064] A further readily identifiable phenotype exhibited by cells of the present composition is the ability to restore the proteolytic balance, the MMP-TIMP balance, the ability to decrease expression of MMPs relative to the expression of TIMPs, or the ability to increase expression of TIMPs relative to expression of

MMPs. Proteolytic balance activity can be determined using an in vitro TIMP assay and/or an in vitro MMP assay described below. [0065] In a typical operative embodiment of the present invention, cells need not exhibit more than one of the foregoing phenotypes. In certain embodiments, cells can exhibit more than one of the foregoing phenotypes.

[0066] While the foregoing phenotypes each typify a functional endothelial cell, a non-endothelial cell exhibiting such a phenotype(s) is considered endothelial-like for purposes of the present invention and thus suitable for use with the present invention. Cells that are endothelial-like are also referred to herein as functional analogs of endothelial cells; or functional mimics of endothelial cells. Thus, by way of example only, cells suitable for use with the materials and methods disclosed herein also include stem cells or progenitor cells that give rise to endothelial-like cells; cells that are non-endothelial cells in origin yet perform functionally like an endothelial cell using the parameters set forth herein; cells of any origin which are engineered or otherwise modified to have endothelial-like functionality using the parameters set forth herein. [0067] Typically, cells of the present invention exhibit one or more of the aforementioned phenotypes when present in confluent, near-confluent or post- confluent populations and associated with a preferred biocompatible matrix such as those described elsewhere herein. As will be appreciated by one of ordinary skill in the art, confluent, near-confluent or post-confluent populations of cells are identifiable readily by a variety of techniques, the most common and widely- accepted of which is direct microscopic examination. Others include evaluation of cell number per surface area using standard cell counting techniques such as but not limited to a hemacytometer or coulter counter. [0068] Additionally, for purposes of the present invention, endothelial-like cells include but are not limited to cells which emulate or mimic functionally and phenotypcially confluent, near-confluent or post-confluent endothelial cells as measured by the parameters set forth herein. [0069] Thus, using the detailed description and guidance set forth below, the practitioner of ordinary skill in the art will appreciate how to make, use, test and identify operative embodiments of the implantable material disclosed herein. That is, the teachings provided herein disclose all that is necessary to make and use the present invention's implantable materials. And further, the teachings provided herein disclose all that is necessary to identify, make and use operatively equivalent cell-containing compositions. At bottom, all that is required is that equivalent cell- containing compositions are effective to treat tubular and non-tubular tissue, structure or organs in accordance with the methods disclosed herein. As will be appreciated by the skilled practitioner, equivalent embodiments of the present composition can be identified using only routine experimentation together with the teachings provided herein.

[0070] In certain preferred embodiments, endothelial cells used in the implantable material of the present invention are isolated from the aorta of human cadaver donors. Each lot of cells is derived from a single or multiple donors, tested extensively for endothelial cell purity, biological function, the presence of bacteria, fungi, known human pathogens and other adventitious agents. The cells are cryopreserved and banked using well-known techniques for later expansion in culture for subsequent formulation in biocompatible implantable materials. [0071] Cell Preparation. As stated above, suitable cells can be obtained from a variety of tissue types and cell types. In certain preferred embodiments, human aortic endothelial cells used in the implantable material are isolated from the aorta of cadaver donors. In other embodiments, porcine aortic endothelial cells are isolated from normal porcine aorta by a similar procedure used to isolate human aortic endothelial cells. Each lot of cells is derived from a single or multiple donors, tested extensively for endothelial cell viability, purity, biological function, the presence of mycoplasma, bacteria, fungi, yeast, known human pathogens and other adventitious agents. The cells are further expanded, characterized and cryopreserved to form a working cell bank at the third to sixth passage using well-known techniques for later expansion in culture and for subsequent formulation in biocompatible implantable material.

[0072] The human or porcine aortic endothelial cells are prepared in T-75 flasks pre-treated by the addition of approximately 15 ml of endothelial cell growth media per flask. Human aortic endothelial cells are prepared in Endothelial Growth Media (EGM-2, Lonza Biosciences, Basel, Switzerland). EGM-2 consists of Endothelial Cell Basal Media (EBM-2, Lonza Biosciences) supplemented with EGM-2 singlequots, which contain 2% FBS. Porcine cells are prepared in EBM-2 supplemented with 5% FBS and 50 μg/ml gentamicin. The flasks are placed in an incubator maintained at approximately 37°C and 5% CO₂ / 95% air, 90% humidity for a minimum of 30 minutes. One or two vials of the cells are removed from the - 16O⁰C to -140°C freezer and thawed at approximately 37⁰C. Each vial of thawed cells is seeded into two T-75 flasks at a density of approximately 3 x 10³ cells per cm , preferably, but no less than 1.0 x 10³ and no more than 7.0 x 10³; and the flasks containing the cells are returned to the incubator. After about 8-24 hours, the spent media is removed and replaced with fresh media. The media is changed every two to three days, thereafter, until the cells reach approximately 85-100% confluence preferably, but no less than 60% and no more than 100%. When the implantable material is intended for clinical application, only antibiotic-free media is used in the post-thaw culture of human aortic endothelial cells and manufacture of the implantable material of the present invention.

[0073] The endothelial cell growth media is then removed, and the monolayer of cells is rinsed with 10 ml of HEPES buffered saline (HEPES). The HEPES is removed, and 2 ml of trypsin is added to detach the cells from the surface of the T- 75 flask. Once detachment has occurred, 3 ml of trypsin neutralizing solution (TNS) is added to stop the enzymatic reaction. An additional 5 ml of HEPES is added, and the cells are enumerated using a hemocytometer. The cell suspension is centrifuged and adjusted to a density of, in the case of human cells, approximately 1.75 x 10⁶ cells/ml using EGM-2 without antibiotics, or in the case of porcine cells, approximately 1.5O x 10 cells/ml using EBM-2 supplemented with 5% FBS and 50 μg/ml gentamicin.

[0074] Biocompatible Matrix. According to the present invention, the implantable material comprises a biocompatible matrix. The matrix is permissive for cell growth and attachment to, on or within the matrix. The matrix is flexible and conformable. The matrix can be a solid, a semi-solid or flowable porous composition. For purposes of the present invention, flowable composition means a composition susceptible to administration using an injection or injection-type delivery device such as, but not limited to, a needle, a syringe or a catheter. Other delivery devices which employ extrusion, ejection or expulsion are also contemplated herein. Porous matrices are preferred. A preferred flowable composition is shape-retaining. The matrix also can be in the form of a flexible planar form. The matrix also can 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 has a particulate form.

[0075] The matrix, when implanted at or adjacent to a tissue, structure or organ can reside at the implantation site for at least about 56-84 days, preferably about at least 7 days, more preferably about at least 14 days, most preferably about at least 28 days before it bioerodes.

[0076] One preferred matrix is Gelfoam^® (Pfizer, New York, NY), an absorbable gelatin sponge (hereinafter "Gelfoam matrix"). Another preferred matrix is Surgifoam (Johnson & Johnson, New Brunswick, NJ), also an absorbable gelatin sponge. Gelfoam and Surgifoam matrices are porous and flexible surgical sponges prepared from a specially treated, purified porcine dermal gelatin solution.

[0077] According to another embodiment, the biocompatible matrix material can be a modified matrix material. Modifications to the matrix material can be selected to optimize and/or to control function of the cells, including the cells' phenotype (e.g., the inhibitory phenotype) as described above, when the cells are associated with the matrix. According to one embodiment, modifications to the matrix material include coating the matrix with attachment factors or adhesion peptides that enhance the ability of the cells to inhibit fibroblast proliferation and migration, to decrease abnormal collagen deposition, to decrease fibrosis, to decrease tissue thickening, to increase TIMP production, to increase NO production, and/or to increase TGF-Bi production. Exemplary attachment factors include, for example, fibronectin, fibrin gel, and covalently attached cell adhesion ligands (including for example RGD) utilizing standard aqueous carbodiimide chemistry. Additional cell adhesion ligands include peptides having cell adhesion recognition sequences, including but not limited to: RGDY, REDVY, GRGDF, GPDSGR, GRGDY and REDV.

[0078] According to another embodiment, the matrix is a matrix other than Gelfoam. Additional exemplary matrix materials include, for example, fibrin gel, alginate, polystyrene sodium sulfonate microcarriers, collagen coated dextran microcarriers, PLA/PGA and pHEMA/MMA copolymers (with polymer ratios ranging from 1-100% for each copolymer). According to one embodiment, a synthetic matrix material, for example, PLA/PGA, is treated with NaOH to increase the hydrophilicity of the material and, therefore, the ability of the cells to attach to the material. According to a preferred embodiment, these additional matrices are modified to include attachment factors or adhesion peptides, as recited and described above. Exemplary attachment factors include, for example, gelatin, collagen, fibronectin, fibrin gel, and covalently attached cell adhesion ligands (including RGD) utilizing standard aqueous carbodiimide chemistry. Additional cell adhesion ligands include peptides having cell adhesion recognition sequences, including but not limited to: RGDY, REDVY, GRGDF, GPDSGR, GRGDY and REDV. [0079] According to another embodiment, the biocompatible matrix material is physically modified to improve cell attachment to the matrix. According to one embodiment, the matrix is cross linked to enhance its mechanical properties and to improve its cell attachment and growth properties. According to a preferred embodiment, an alginate matrix is first cross linked using calcium sulfate followed by a second cross linking step using calcium chloride and routine protocols. [0080] According to yet another embodiment, the pore size of the biocompatible matrix is modified. A preferred matrix pore size 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 μm to 100 μm. Other preferred pore sizes include pore sizes below about 25 μm and above about 100 μm. According to one embodiment, the pore size 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/salt block is then immersed in water and the salt leached out leaving a porous structure. The solvent is chosen so that the matrix is in the solution but the salt is not. One exemplary solution includes PLA and methylene chloride. [0081] 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 surfactant. The gas bubbles are subsequently removed using a vacuum, leaving a porous structure.

[0082] According to another embodiment, a freeze-drying technique is employed to control the pore size of the matrix, using the freezing rate of the ice microparticles to form pores of different sizes. For example, a gelatin solution of about 0.1-2% porcine or bovine gelatin 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 cross-linked by using, preferably, ultraviolet light (254 nm) or by adding gluteraldehyde (formaldehyde). Variations in pre-freezing temperature (for example -2O⁰C, -8O⁰C or -180⁰C), lyophilizing temperature (freeze dry at about -50⁰C), and gelatin concentration (0.1% to 2.0%; pore size is generally inversely proportional to the concentration of gelatin in the solution) can all affect the resulting pore size of the matrix material and can be modified to create a preferred material. The skilled artisan will appreciate that a suitable pore size is that which promotes and sustains optimal cell populations having the phenotypes described elsewhere herein.

[0083] Flexible Planar Form. As taught herein, planar forms of biocompatible matrix can be configured in a variety of shapes and sizes, preferably a shape and size which is adapted for implantation at, adjacent or in the vicinity of a tubular or non- tubular tissue, structure or organ, for example, in the case of a vascular access structure such as a fistula, graft, peripheral graft, or other vascular access structure and its surrounds, a shape and size which can conform to the contoured surfaces of the access structure and its associated blood vessels. According to a preferred embodiment, a single piece of matrix is sized and configured for application to the specific tubular or non-tubular tissue, structure or organ to be treated. [0084] According to one embodiment, the biocompatible matrix is configured as a flexible planar form. Exemplary embodiments configured for administration to a tubular tissue, structure or organ, such as but not limited to a blood vessel, and considerations for design and administration of the flexible planar form of the biocompatible matrix are described in greater detail in co-pending application PCT/US05/43967 filed on December 6, 2005 (also know as Attorney Docket No. ELV-002PC), the teachings of which are incorporated by reference herein in their entirety. [0085] In part, the invention disclosed herein is based on the discovery that a contoured and/or conformable flexible planar form allows the implantable material to be applied optimally to a tubular or non-tubular tissue, structure or organ without compromising the integrity of the implant or the cells engrafted thereto. One preferred embodiment optimizes contact with and conforms to the anatomy of the tissue, structure or organ and controls the extent of overlap of implantable material. Excessive overlap of implantable material within the space can cause pressure points on the treated tissue, structure or organ, potentially restricting fluid flow to or through the tissue or creating other disruptions that could delay and/or inhibit homeostasis and normal healing. The skilled practitioner will recognize excessive overlap at the time of implantation and will recognize the need to reposition or alter, e.g., trim, the implantable material. Additionally, in other embodiments, overlap of implantable material can result in over-dosing of therapeutic agents dispersed within the implantable material. As described elsewhere herein, chemicals or other exogenously supplied therapeutic agents can be optionally added to an implant. In certain other embodiments, such agents can be added to a biocompatible matrix and administered in the absence of cells; a biocompatible matrix used in this manner optionally defines a slot. [0086] In contrast, implantable material that does not adequately contact the target tubular structure can lead to insufficient exposure to the clinical benefits provided by the engrafted cells or an under-dosing of therapeutic agent added to the implantable material. The skilled practitioner will recognize that sub-optimal contact at the time of implantation necessitates re-positioning and/or additional implantable material. [0087] Flowable Composition. In certain embodiments contemplated herein, the implantable material of the present invention is a flowable composition comprising a particulate biocompatible matrix. Any non-solid flowable composition for use with an injectable-type delivery device capable of either intraluminal administration by navigating the interior length of a tubular structure such as a blood vessel or by percutaneous local administration is contemplated herein. The flowable composition is preferably a shape-retaining composition. Thus, an implantable material comprising cells in, on or within a flowable-type particulate matrix as contemplated herein can be formulated for use with any injectable delivery device ranging in internal diameter from about 22 gauge to about 26 gauge and capable of delivering about 50 mg of flowable composition comprising particulate material containing preferably about 1 million cells in about 1 to about 3 ml. [0088] According to a currently preferred embodiment, the flowable composition comprises a biocompatible particulate matrix such as Gelfoam^® particles, Gelfoam^® powder, or pulverized Gelfoam^® (Pfizer Inc., New York, NY) (hereinafter "Gelfoam particles"), a product derived from porcine dermal gelatin. According to another embodiment, the particulate matrix is Cytodex-3 (Amersham Biosciences, Piscataway, NJ) microcarriers, comprised of denatured collagen coupled to a matrix of cross-linked dextran. According to a further embodiment, the particulate matrix is CultiSpher-G (Percell Biolytica AB, Astorp, Sweden) microcarrier, comprised of porcine gelatin. According to another embodiment, the particulate matrix is a macroporous material. According to one embodiment, the macroporous particulate matrix is CytoPore (Amersham Biosciences, Piscataway, NJ) microcarrier, comprised of cross-linked cellulose which is substituted with positively charged N,N,-diethylaminoethyl groups.

[0089] According to alternative embodiments, the biocompatible implantable particulate matrix is a modified biocompatible matrix. Modifications include those described above for an implantable matrix material.

[0090] Examples of flowable compositions suitable for use in this manner are disclosed in application PCT/US05/44090 filed on December 6, 2005 (also known as Attorney Docket No. ELV-008PC), the entire contents of which is herein incorporated by reference; and, application PCT/US05/43844 filed on December 6, 2005 (also known as Attorney Docket No. ELV-009PC), the entire contents of which are herein incorporated by reference.

[0091] Cell Seeding of Biocompatible Matrix. Pre-cut pieces of a suitable biocompatible matrix or an aliquot of suitable biocompatible flowable matrix are re- hydrated by the addition of EGM-2 without antibiotics at approximately 37⁰C and 5% CO₂ / 95% air for 12 to 24 hours. The implantable material is then removed from their re-hydration containers and placed in individual tissue culture dishes. Biocompatible matrix is seeded at a preferred density of approximately 1.5-2.0 x 10³ cells (1.25-1.66 x 10⁵ cells /cm of matrix) and placed in an incubator maintained at approximately 37⁰C and 5% CO₂ / 95% air, 90% humidity for 3-4 hours to facilitate cell attachment. According to one embodiment, the seeded matrix is then placed into individual containers (Evergreen, Los Angeles, CA) tubes, each fitted with a cap containing a 0.2 μm filter with EGM-2 and incubated at approximately 37⁰C and 5% CO₂ / 95% air. According to an alternative embodiment, three seeded matrices are placed into a single container tube for incubation. The media is changed every two to three days, thereafter, until the cells have reached confluence. The cells in one preferred embodiment are preferably passage 6, but cells of fewer or more passages can be used. Further implantable material preparation protocols according to additional embodiments of the invention are disclosed in co-pending application PCT/US05/43844 filed on December 6, 2005 (also known as Attorney Docket No. ELV-009PC), the entire contents of which are herein incorporated by reference. [0092] Cell Growth Curve and Confluence. 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 viability, and a growth curve is constructed and evaluated in order to assess the growth characteristics and to determine whether confluence, near- confluence or post-confluence has been achieved. Representative growth curves from two preparations of implantable material comprising porcine aortic endothelial cell implanted lots are presented in FIGS. IA and IB. In these examples, the implantable material is in a flexible planar form. Generally, one of ordinary skill will appreciate the indicia of acceptable cell growth at early, mid- and late time points, such as observation of an increase in cell number at the early time points (when referring to FIG. IA, between about days 2-6), followed by a near confluent phase (when referring to FIG. IA, between about days 6-8), followed by a plateau in cell number once the cells have reached confluence (when referring to FIG. IA, between about days 8-10) and maintenance of the cell number when the cells are post-confluent (when referring to FIG. IA, between about days 10-14). For purposes of the present invention, cell populations which are in a plateau for at least 72 hours are preferred. [0093] Cell counts are achieved by complete digestion of the aliquot of implantable material with a solution of 0.5 mg/ml collagenase in a CaCL₂ solution. After measuring the volume of the digested implantable material, a known volume of the cell suspension is diluted with 0.4% trypan blue (4: 1 cells to trypan blue) and viability assessed by trypan blue exclusion. Viable, non-viable and total cells are enumerated using a hemacytometer. Growth curves are constructed by plotting the number of viable cells versus the number of days in culture. Cells are shipped and implanted after reaching confluence. [0094] For purposes of the present invention, confluence is defined as the presence of at least about 4 x 10⁵ cells/cm³ when in a flexible planar form of the implantable material (1.0 x 4.0 x 0.3 cm), and preferably about 7 x 10⁵ to 1 x 10⁶ total cells per aliquot (50-70 mg) when in the flexible composition. For both, cell viability is at least about 90% preferably but no less than 80%. If the cells are not confluent by day 12 or 13, the media is changed, and incubation is continued for an additional day. This process is continued until confluence is achieved or until 14 days post-seeding. On day 14, if the cells are not confluent, the lot is discarded. If the cells are determined to be confluent after performing in-process checks, a final media change is performed. This final media change is performed using EGM-2 without phenol red and without antibiotics. Immediately following the media change, the tubes are fitted with sterile plug seal caps for shipping.

[0095] Evaluation of Functionality. For purposes of the invention described herein, the implantable material is further tested for indicia of functionality prior to implantation. For example, conditioned media are collected during the culture period to ascertain levels of heparan sulfate (HS), transforming growth factor-βi (TGF-βi), basic fibroblast growth factor (b-FGF), tissue inhibitors of matrix metalloproteinases (TIMP), and nitric oxide which are produced by the cultured endothelial cells. In certain preferred embodiments, the implantable material can be used for the purposes described herein when total cell number is at least about 2, preferably at least about 4 x 10⁵ cells/cm³ of flexible planar form; percentage of viable cells is at least about 80-90%, preferably >90%, most preferably at least about 90%; heparan sulfate in conditioned media is at least about 0.1-0.5, preferably at least about 0.23 microg/mL/day; TGF-βi in conditioned media is at least about 200- 300, preferably at least about 300 picog/ml/day; b-FGF in conditioned media is below about 200 picog/ml, preferably no more than about 400 picog/ml; TIMP-2 in conditioned media is at least about 5.0 - 10.0 ng/mL/day, preferably at least about 8.0 ng/mL/day; NO in conditioned media is at least about 0.5 - 3.0 μmol/L/day, preferably at least about 2.0 μmol/L/day. [0096] Heparan sulfate levels can be quantitated using a routine dimethylmethylene blue-chondroitinase ABC digestion spectrophotometric assay. Total sulfated glycosaminoglycan (GAG) levels are determined using a dimethylmethylene blue (DMB) dye binding assay in which unknown samples are compared to a standard curve generated using known quantities of purified chondroitin sulfate diluted in collection media. Additional samples of conditioned medium are mixed with chondroitinase ABC to digest chondroitin and dermatan sulfates prior to the addition of the DMB color reagent. All absorbances are determined at the maximum wavelength absorbance of the DMB dye mixed with the GAG standard, generally around 515-525 nm. The concentration of heparan sulfate per day is calculated by subtracting the concentration of chondroitin and dermatan sulfate from the total sulfated glycosaminoglycan concentration in conditioned medium samples. Chondroitinase ABC activity is confirmed by digesting a sample of purified chondroitin sulfate. Conditioned medium samples are corrected appropriately if less than 100% of the purified chondroitin sulfate is digested. Heparan sulfate levels may also be quantitated using an ELISA assay employing monoclonal antibodies.

[0097] TGF-βι_, TIMP, and b-FGF levels can be quantitated using an ELISA assay employing monoclonal or polyclonal antibodies, preferably polyclonal. Control collection media can also be quantitated using an ELISA assay and the samples corrected appropriately for TGF-Pi₁ TIMP, and b-FGF levels present in control media.

[0098] Nitric oxide (NO) levels can be quantitated using a standard Griess Reaction assay. The transient and volatile nature of nitric oxide makes it unsuitable for most detection methods. However, two stable breakdown products of nitric oxide, nitrate (NO₃) and nitrite (NO₂), can be detected using routine photometric methods. The Griess Reaction assay enzymatically converts nitrate to nitrite in the presence of nitrate reductase. Nitrite is detected colorimetrically as a colored azo dye product, absorbing visible light in the range of about 540 nm. The level of nitric oxide present in the system is determined by converting all nitrate into nitrite, determining the total concentration of nitrite in the unknown samples, and then comparing the resulting concentration of nitrite to a standard curve generated using known quantities of nitrate converted to nitrite. [0099] The earlier-described preferred inhibitory phenotype is assessed using the quantitative heparan sulfate, TGF-Bi₁ TIMP, NO and/or b-FGF assays described above, as well as quantitative in vitro assays of smooth muscle cell growth and platelet aggregation as follows. For purposes of the present invention, implantable material is ready for implantation when one or more of these alternative in vitro assays confirm that the implantable material is exhibiting the preferred inhibitory phenotype.

[00100] To evaluate inhibition of smooth muscle cell growth in vitro, the magnitude of inhibition associated with cultured endothelial cells is determined. Porcine or human aortic smooth muscle cells are sparsely seeded in 24 or 96 well tissue culture plates in smooth muscle cell growth medium (SmGM-2, Lonza BioScience). The cells are allowed to attach for 24 hours. The medium is then replaced with smooth muscle cell basal media (SmBM) containing 0.2% FBS for 48-72 hours to growth arrest the cells. Conditioned media is prepared from post- confluent endothelial cell cultures, diluted 1 : 1 with 2X SMC growth media and added to the cultures. A positive control for inhibition of smooth muscle cell growth is included in each assay. After three to four days, the number of cells in each sample is enumerated using a Coulter Counter or a colorimetric assay after the addition of a dye. The effect of conditioned media on smooth muscle cell proliferation is determined by comparing the number of smooth muscle cells per well immediately before the addition of conditioned medium with that after three to four days of exposure to conditioned medium, and to control media (standard growth media with and without the addition of growth factors). The magnitude of inhibition associated with the conditioned media samples are compared to the magnitude of inhibition associated with the positive control. According to a preferred embodiment, the implantable material is considered inhibitory if the conditioned media inhibits about 20% of what the heparin control is able to inhibit. [00101] To evaluate inhibition of fibroblast collagen deposition activity in vitro, the magnitude of inhibition associated with cultured endothelial cells is determined. Porcine or human aortic fibroblasts are sparsely seeded in 24 or 96 well tissue culture plates in smooth muscle cells growth medium (SmGM-2, Lonza BioScience) supplemented with growth factors to activate the fibroblasts to secrete and deposit collagen. The cells are allowed to attach for 24 hours. The medium is then replaced with smooth muscle cell basal media (SmBM) containing 0.2% FBS for 48-72 hours to growth arrest the cells. Conditioned media is prepared from post-confluent endothelial cell cultures, diluted 1 : 1 with 2X SMC growth media and added to the cultures. A positive control for inhibition of fibroblast collagen deposition activity is included in each assay. After three to four days, the number of cells in each sample is enumerated using a Coulter Counter. The effect of conditioned media on fibroblast collagen deposition activity is determined by comparing the amount of collagen deposition per well immediately before the addition of conditioned medium with that after three to four days of exposure to conditioned medium, and to control media (standard growth media with and without the addition of growth factors). The magnitude of inhibition associated with the conditioned media samples are compared to the magnitude of inhibition associated with the positive control. According to a preferred embodiment, the implantable material is considered inhibitory if the conditioned media inhibits about 20% of what the heparin control is able to inhibit. [0100] To evaluate inhibition of thrombosis in vitro, the level of heparan sulfate associated with the cultured endothelial cells is determined. Heparan sulfate has both anti-proliferative and anti-thrombotic properties. Using either the routine dimethylmethylene blue-chondroitinase ABC spectrophotometric assay or an ELISA assay, both assays are described in detail above, the concentration of heparan sulfate is calculated. The implantable material can be used for the purposes described herein when the heparan sulfate in the conditioned media is at least about 0.23-1.0, preferably at least about 0.5 microg/mL/day. [0101] Another method to evaluate inhibition of thrombosis involves determining the magnitude of inhibition of platelet aggregation in vitro associated with platelet rich-plasma or platelet concentration (Research Blood Components, Brighton, MA). Conditioned media is prepared from post-confluent endothelial cell cultures and added to aliquots of the platelet concentrate. A platelet aggregating agent (agonist) is added to the platelets seeded into 96 well plates as control. Platelet agonists commonly include arachidonate, ADP, collagen type I, epinephrine, thrombin (Sigma-Aldrich Co., St. Louis, MO) or ristocetin (available from Sigma- Aldrich Co., St. Louis, MO). An additional well of platelets has no platelet agonist or conditioned media added, to assess for baseline spontaneous platelet aggregation. A positive control for inhibition of platelet aggregation is also included in each assay. Exemplary positive controls include aspirin, heparin, indomethacin (Sigma- Aldrich Co., St. Louis, MO), abciximab (ReoPro®, Eli Lilly, Indianapolis, IN), tirofiban (Aggrastat®, Merck & Co., Inc., Whitehouse Station, NJ) or eptifibatide (Integrilin®, Millennium Pharmaceuticals, Inc., Cambridge, MA). The resulting platelet aggregation of all test conditions are then measured using a plate reader and the absorbance read to 405 nm. The platelet reader measures platelet aggregation by monitoring optical density. As platelets aggregate, more light can pass through the specimen. The platelet reader reports results in absorbance, a function of the rate at which platelets aggregate. Aggregation is assessed as maximal aggregation between 6-12 minutes after the addition of the agonist. The effect of conditioned media on platelet aggregation is determined by comparing maximal agonist aggregation before the addition of conditioned medium with that after exposure of platelet concentrate to conditioned medium, and to the positive control. Results are expressed as a percentage of the baseline. The magnitude of inhibition associated with the conditioned media samples are compared to the magnitude of inhibition associated with the positive control. According to a preferred embodiment, the implantable material is considered regulatory if the conditioned media inhibits thrombosis by at least about 20% of the control, more preferably by at least about 40% of the control, and most preferably by at least about 60% of the control. [0102] When ready for implantation, the implantable material comprising a flexible planar form is supplied in final product containers, each preferably containing a 1 x 4 x 0.3 cm (1.2 cm³) sterile piece with preferably approximately 5-8 x 10⁵ preferably at least about 4 x 10⁵ cells/cm³ and at least about 90% viable cells, for example, human aortic endothelial cells derived from a single cadaver donor source, per cubic centimeter in approximately 45-60 ml, preferably about 50 ml, endothelial growth medium (for example, endothelial growth medium (EGM-2) containing no phenol red and no antibiotics). According to an alternative embodiment, the final product container contains three sterile pieces in approximately 100 - 200 ml, preferably about 150 ml, endothelial growth medium (for example, endothelial growth medium (EGM-2) containing no phenol red and no antibiotics). When porcine aortic endothelial cells are used, the storage and transport medium is also EBM-2 containing no phenol red, but supplemented with 5% FBS, and 50 μg/ml gentamicin. [0103] In other preferred embodiments, implantable material comprising a flowable particulate form is supplied in final product containers, including, for example, sealed tissue culture containers modified with filter caps or pre-loaded syringes. According to one embodiment, each container preferably contains about 50-60 mg of particulate microparticle material engrafted with about 7 x 10⁵ to about 1 x 10 total endothelial cells in about 45-60 ml, preferably about 50 ml, transport medium per aliquot. According to another embodiment, each container preferably contains about 5 - 15 mg of macroporous bead material engrafted with about 1.25 x 10⁶ to about 3.75 x 10⁶ total endothelial cells in about 10 - 50 ml, preferably about 20 ml, transport medium per aliquot. [0104] Shelf-Life of Implantable Material. The implantable material comprising a confluent, near-confluent or post-confluent population of cells can be maintained at room temperature in a stable and viable condition for at least two weeks. Preferably, such implantable material is maintained in about 45-60 ml, more preferably 50 ml per implantable material, transport media with or without additional FBS or VEGF. Transport media comprises EGM-2 media without phenol red. According to one embodiment, VEGF can be added to the volume of transport media up to a total VEGF concentration of about 3-4 ng/ml. According to another embodiment, FBS can be added to the volume of transport media with an additional about 8-10% FBS, bringing the total concentration of FBS in transport media to about 10-12%. However, because FBS must be removed from the implantable material prior to implantation, it is preferred to limit the amount of FBS used in the transport media to reduce the length of rinse required prior to implantation. [0105] Cryopreservation of Implantable Material. The confluent implantable material comprising confluent population of cells can be cryopreserved for storage and/or transport to the clinic without diminishing its clinical potency or integrity upon eventual thaw. Preferably, the implantable material is cryopreserved in a 15 ml cryovial (Nalgene®, Nalge Nunc Int'l, Rochester, NY) in a solution of about 5 ml CryoStor CS-IO solution (BioLife Solutions, Oswego, NY) containing about 10% DMSO, about 2-8% Dextran and about 20-75% FBS and/or human serum. Cryovials are placed in a cold iso-propanol water bath, transferred to an -8O⁰C freezer for 4 hours, and subsequently transferred to liquid nitrogen (-150 to -165⁰C). [0106] Cryopreserved aliquots of the implantable material are then slowly thawed at room temperature for about 15 minutes, followed by an additional approximately 15 minutes in a room temperature water bath. The material is then washed about 3 times in about 200-250 niL saline, lactated ringers, or EBM solution. The three rinse procedures are conducted for about 5 minutes at room temperature. If the aliquot of the implantable material is intended for in vivo implantation into a subject, the implantable material is considered ready for implantation following the three rinse procedures.

[0107] If the aliquot of the implantable material is intended for in vitro evaluation, following the thaw and rinse procedures, the cryopreserved material is allowed to rest for about 48 hours in about 10 ml of recovery solution. For porcine endothelial cells, the recovery solution is EBM-2 supplemented with 5% FBS and 50 μg/ml gentamicin at 37°C in 5% CO2. For human endothelial cells, the recovery solution is EGM-2 with or without antibiotics. Further post-thaw conditioning can be carried out for at least another 24 hours prior to use and/or packaging for storage or transport. [0108] Immediately prior to implantation, the transport or cryopreservation medium is decanted and implantable material is rinsed in about 250-500 ml sterile saline (USP). The medium in the final product contains a small amount of FBS to maintain cell viability during transport to a clinical site if necessary. The FBS has been tested extensively for the presence of bacteria, fungi and other viral agents according to Title 9 CFR: Animal and Animal Products. A rinsing procedure is employed just prior to implantation, which decreases the amount of FBS transferred preferably to between 0-60 ng per implant, but preferably no more than 1 -2 μg per implant.

[0109] The total cell load per human patient will be preferably approximately 1.6-2.6 x 10⁴ cells per kg body weight, but no less than about 2 x 10³ and no more than about 2 x 10⁶ cells per kg body weight.

[0110] As contemplated herein, the implantable material of the present invention comprises cells, preferably vascular endothelial cells, which are preferably about 90% viable at a density of preferably about 4 x 10⁵ cells/cm³ of flexible planar form, and when confluent, produce conditioned media containing heparan sulfate at at least about 0.1-0.5, preferably at least about 0.23 microg/mL/day; TGF-βi in conditioned media is at least about 200-300, preferably at least about 300 picog/ml/day; b-FGF in conditioned media is below about 200 picog/ml, preferably no more than about 400 picog/ml; TIMP-2 in conditioned media is at least about 5.0 - 10.0 ng/mL/day, preferably at least about 8.0 ng/mL/day; NO in conditioned media is at least about 0.5 - 3.0 μmol/L/day, preferably at least about 2.0 μmol/L/day. Delivery of Implantable Material in Flexible Planar Form

[0111] General Considerations. The implantable material can be administered to a tubular or non-tubular tissue, structure or organ in a variety of forms. According to one preferred embodiment, the implantable material is a flexible planar form cut in a shape and size which is adapted for implantation adjacent to a tubular or non- tubular tissue, structure or organ and its surrounds and which can conform to the contoured surfaces of the tissue, structure or organ and its surrounds. [0112] According to a preferred embodiment, a single piece of implantable material is sized for application to the tubular or non-tubular tissue, structure or organ to be treated. According to another embodiment, more than one piece of implantable material in its flexible planar form, for example, two, three, four, five, six, seven, eight or more pieces of matrix material, can be applied to a single treatment location. Additionally, more than one location along the length of a tubular or non-tubular tissue, structure or organ can be treated with one or more pieces of the implantable material. For example, in the case of the creation of an arteriovenous graft, each of the venous and arterial anastomosis and the distal venous segment can be treated with one or more pieces of the implantable matrix material.

[0113] According to one non-limiting embodiment, the implantable material is configured to conform to an exterior surface of a tubular or non-tubular tissue, structure or organ. An exemplary non-limiting planar form has a length of about 2 cm to about 6 cm, a width of about 0.5 cm to about 2 cm, and a height of about 0.1 cm to about 0.5 cm. According to another embodiment, the flexible planar form can be configured as an anatomically contoured form which conforms to an exterior surface of a tubular or non-tubular tissue, structure or organ. Exemplary flexible planar forms configured for administration to a vascular structure are discussed in greater detail in International application PCT/US05/43967 filed on December 6, 2005 (also known as Attorney Docket No. ELV-002PC). Delivery of Implantable Material in a Flowable Composition

[0114] General Considerations. The implantable material of the present invention when in a flowable composition comprises a particulate biocompatible microporous or macroporous matrix and cells, preferably endothelial cells, more preferably vascular endothelial cells, which are about 90% viable at a preferred density of about 0.8 x 10⁴ cells/mg, more preferred of about 1.5 x 10⁴ cells/mg, most preferred of about 2 x 10⁴ cells/mg in microporous matrix particles, a preferred density of about 0.5 x 10⁵ cells/mg, more preferred of about 1.0 x 10 cells/mg, most preferred of about 2.5 x 10⁵ cells/mg in macroporous matrix particles, and which can produce conditioned media containing heparan sulfate at at least about 0.1-0.5, preferably at least about 0.23 microg/mL/day; TGF-βi in conditioned media is at least about 200-300, preferably at least about 300 picog/ml/day; b-FGF in conditioned media is below about 200 picog/ml, preferably no more than about 400 picog/ml; TIMP-2 in conditioned media is at least about 5.0 - 10.0 ng/mL/day, preferably at least about 8.0 ng/mL/day; NO in conditioned media is at least about 0.5 - 3.0 μmol/L/day, preferably at least about 2.0 μmol/L/day; and, display the earlier-described inhibitory phenotype. [0115] For purposes of the present invention generally, administration of the flowable particulate material is localized to a site at, adjacent to or in the vicinity of the tubular or non-tubular tissue, structure or organ to be treated. As contemplated herein, localized deposition can be accomplished as follows. [0116] In a particularly preferred embodiment, the flowable composition is first administered percutaneously, entering the space adjacent to the tissue, structure or organ to be treated and then deposited on at, into or adjacent to the site of the tissue, structure or organ to be treated using a suitable needle, catheter or other suitable percutaneous injection-type delivery device. Alternatively, the flowable composition is delivered percutaneously using a needle, catheter or other suitable delivery device in conjunction with an identifying step to facilitate delivery to a desired tissue, structure or organ site to be treated. The identifying step can occur prior to or coincident with percutaneous delivery. The identifying step can be accomplished using intravascular ultrasound, other routine ultrasound, fluoroscopy, and/or endoscopy methodologies, to name but a few. The identifying step is optionally performed and not required to practice the methods of the present invention.

[0117] The flowable composition can also be administered intraluminally, for example, endovascularly. For example, the composition can be delivered by any device able to be inserted within a blood vessel or other tubular structure. In this instance, such an intraluminal delivery device is equipped with a traversing or penetrating device which penetrates the luminal wall of a vessel wall or other tubular structure to reach a non-luminal surface of the tubular structure. The flowable composition is then deposited on a non-luminal surface of the tubular structure at, adjacent to, or in the vicinity of the tubular structure or site of intervention of the structure.

[0118] It is contemplated herein that a non-luminal, also termed an extraluminal, surface can include an exterior or perivascular surface of a tubular structure, for example, a blood vessel, or can be within the adventitia, media, or intima of the tubular structure. For purposes of this invention, non-luminal or extraluminal is any surface except an interior surface of the lumen.

[0119] The penetrating devices contemplated herein can permit, for example, a single point of delivery or a plurality of delivery points arranged in a desired geometric configuration to accomplish delivery of flowable composition to a surface of a tubular or non-tubular tissue, structure or organ without disrupting the integrity of the tissue, structure or organ. A plurality of delivery points can be arranged, for example, in a circle, a bulls-eye, or a linear array arrangement to name but a few. [0120] Preferably, the flowable formulation of the implantable material is deposited on an extraluminal or exterior surface of a tubular or non-tubular tissue, structure or organ, either at the site of the tubular or non-tubular tissue, structure or organ to be treated, or adjacent to or in the vicinity of the site of the tubular or non- tubular tissue, structure or organ to be treated. The composition can be deposited in a variety of locations relative to the tubular or non-tubular tissue, structure or organ to be treated, for example, in the case of an arterio-venous anastomosis, at the proximal anastomosis, at the distal anastomosis, adjacent to either anastomosis, for example, upstream of the anastomosis, on the opposing exterior vessel surface from the anastomosis. According to a preferred embodiment, an adjacent site is within about 0 mm to 20 mm of the site of the tubular or non-tubular tissue, structure or organ intervention. In another preferred embodiment, a site is within about 21 mm to 40 mm; in yet another preferred embodiment, a site is within about 41 mm to 60 mm. In another preferred embodiment, a site is within about 61 mm to 100 mm. Alternatively, an adjacent site is any other clinician-determined adjacent location where the deposited composition is capable of exhibiting a desired effect on a tubular or non-tubular tissue, structure or organ in the proximity of the site of intervention.

[0121] In another embodiment, the flowable composition is delivered directly to a surgically-exposed site adjacent to or at or in the vicinity of the site of intervention of the tubular or non-tubular tissue, structure or organ. In this case, delivery is guided and directed by direct observation of the site. Also in this case, delivery can be aided by coincident use of an identifying step as described above. Again, the identifying step is optional.

Examples

Example 1 : Animal Study

[0122] This study exemplifies use of the present invention's materials and methods to modulate abnormal or pathological tissue remodeling and locally direct positive tissue modeling. The experimental model chosen for such exemplification is tissue modeling following vascular injury and trauma induced by surgical intervention. Furthermore, this example provides experimental protocols for testing and using a preferred embodiment of the present invention to reduce or modulate indicia of abnormal tissue remodeling including fibroblast cell proliferation and migration, abnormal collagen deposition, fibrosis, lumen diameter, adventitial thickening and expression levels of MMPs following an intervention to a vascular tubular structure, for example, introduction of an AV graft, in animal test subjects. [0123] Using standard surgical procedures, an AV graft was created between the carotid artery and the jugular vein. Implantable material was then disposed in the perivascular space adjacent to each surgically created AV graft anastomosis; the details of one exemplary procedure are set forth below. As described earlier, the placement and configuration of implantable material can be varied. In this study, the implantable material was in a flexible planar form. [0124] Specifically, the study included 20 porcine test subjects undergoing AV graft surgery. Conventional AV graft surgery procedures were performed according to standard operative techniques. Implantable material was applied to the AV graft anastomoses and surrounds as described below after the graft surgery was completed and flow through the graft was established. [0125] For each test subject undergoing AV graft surgery, one six-millimeter internal diameter PTFE graft was placed between the left common carotid artery and right external jugular vein of the test subject. An oblique end-to-side anastomosis was created at each end of the graft using a running 6-0 prolene suture. All test subjects received intra-operative heparin and administered daily aspirin following surgery. [0126] Ten of the test subjects received implantable material comprising aortic endothelial cells on the day of surgery. Five such implants were applied to each test subject. Two implants were wrapped around each of the two anastomotic sites. In this circumstance, one end of the first piece of implantable material was passed under the anastomotic segment until the middle of the implant was at the point where the vessel and graft meet. The second piece of implantable material was then wrapped in a direction opposite that of the first piece, placed on top of the anastomotic segment and the ends tucked under the anastomosis. Both ends were then wrapped around the suture line keeping the implant centered over the suture line. The ends overlapped minimally to secure the material in place. An additional single implant was placed longitudinally along the length of the proximal venous segment starting at the anastomosis, of each test subject. The implant did not completely wrap around the circumference of the vein. [0127] Ten test subjects received control implants without cells, wrapped around the anastomotic sites and placed on the proximal venous segment of the graft on the day of surgery. The total cell load based on body weight was approximately 2.5 xlO⁵ cells per kg.

[0128] Surgical Procedure. Bilateral 8-cm neck incisions were made over the sternocleidomastoid muscle on each side of the neck. Using these incisions, the left common carotid artery was isolated followed by the right external jugular vein. Approximately 4-8 cm segments of vein and artery were freed from surrounding tissues and all tributaries off the vein were ligated with 3-0 silk sutures. A 6-mm internal diameter PTFE graft (Atrium Medical Corp., Hudson, NH) was tunneled in a subcutaneous tract between the two incisions. The isolated jugular vein was clamped and a 10-mm venotomy was made. The vein was irrigated with heparinized saline solution and an oblique end-to-side anastomosis was made between the vein graft using a running 6-0 prolene suture. The average graft length was 18.6 ± 0.9 cm. Once fashioned, the venous clamp was removed, the graft flushed with heparin-saline solution and re-clamped. The left carotid artery was then clamped and an 8-mm arteriotomy performed. The artery was flushed with heparinized saline solution and an oblique end-to-side anastomosis was made between the artery and graft using 6-0 proline suture. Vascular clamps were removed and flow through the graft was confirmed by the physical palpation of thrill in the graft. Hemostatis of each vascular anastomosis was confirmed and on rare occasion an additional 6-0 prolene suture was placed in an interrupted fashion at the point of anastomotic bleeding.

[0129] Following completion of the anastomoses, the PTFE arteriovenous graft was positioned to prevent kinking. The PTFE arteriovenous graft was percutaneously cannulated with a 23-gauge butterfly needle just distal to the carotid artery-graft anastomosis. To confirm placement, blood was aspirated into the system with a 10 cc syringe. The system was then flushed with 10 cc's of saline. A C-arm fluoroscope was then placed over the neck of the study animal so that the venous-graft anastomosis and the venous outflow tract could be visualized. Under continuous fluoroscopy, 10-15 cc's of iodinated contrast (Renograffin, full strength) was injected. The cine angiography was recorded and stored for comparison to the pre-sacrifice angiogram.

[0130] After completion of the angiography, the anastomotic sites were wrapped in a wet 4"x4" gauze sponge. Pressure was maintained on the anastomotic sites for a period of approximately 5 minutes, before removing the gauze sponges and inspecting the anastomotic sites. If hemostasis had not yet been achieved, as was evidenced by oozing of blood, the site was again wrapped for another 5 minutes. Additional sutures were placed at the discretion of the surgeon if the hemorrhage from the site was severe. Once hemostasis had been achieved, the neck wound was filled with sterile saline and flow probe analysis performed at the distal venous outflow tract using a 6-mm Transonic flow probe. The saline was removed, if necessary, and the anastomoses made as dry as possible and treated with either implantable material comprising aortic endothelial cells or control implants. Sites were not treated with either type of implant until all bleeding had been controlled, flow through the graft confirmed and the area made as dry as possible. When complete, the wound was closed in layers and the animal was allowed to recover from anesthesia. [0131] Heparin was administered prior to surgery as a 100 U/kg bolus injection plus a 35 U/kg/hr continuous infusion and maintained until the end of surgery. Additional bolus doses (100U/kg) were administered, as necessary to maintain ACTs > 200 seconds.

[0132] Graft Patency. AV graft patency was confirmed by access flow measurements using color-flow Doppler ultrasound and Transonic flow probe (Transonic Systems, Inc., Ithaca, NY) immediately after surgery, 3-7 days post surgery and once per week thereafter. Grafts were monitored closely for blood flow. [0133] Pathology Procedures. Animal test subjects were anesthetized using sodium pentobarbital (65mg/kg, IV). Graft patency was determined prior to necropsy by cine angiography as described above. After completion of the angiography, the grafts/anastomoses were perfused with PBS followed by formalin. Late venous luminal gain was determined by comparison of the venous lumen diameter post-surgery to that just prior to the 1 month sacrifice. Luminal gain was calculated by dividing each venous diameter by the reference graft diameter using the following equation: (late venous luminal gain) = (venous lumen diameter at 28 days / reference graft diameter) - (venous lumen diameter post-surgery / reference graft diameter).

[0134] Histology. Half of the animal test subjects (5 cell engrafted implant subjects; 5 control implant subjects) were euthanized 3 days following surgery. The remaining animal test subjects (5 cell engrafted implant subjects; 5 control implant subjects) were euthanized one month following surgery. [0135] A limited necropsy, defined as the macroscopic examination of the administration site, including all anastomotic and proximal venous sites, and surrounding tissue including draining lymph nodes was performed on all test subjects. Tissue from major organs, including brain, lungs, kidneys, liver, heart and spleen, were collected and saved for all test subjects euthanized at one month following surgery. The organs were to be analyzed only if unusual findings arose from macroscopic examination of the external surface of the body or from the microscopic examination of administration sites and surrounding tissue. No unusual findings arose that warranted further examination of the major organs in any of the animals enrolled into the study.

[0136] All AV graft anastomotic sites and surrounding tissues, including 5-cm segments each of the anastomosed vein and artery, were trimmed, fixed in 10% formalin (or equivalent) and embedded in glycolmethacrylate (or equivalent). Using approximately 3 μm -thick sections cut with a C-profile stainless steel knife (or equivalent), sections were prepared from at least three regions: the vein graft anastomosis, the graft-artery anastomosis, and the venous outflow tract. Three sections were made transversely through the vein graft anastomosis. Five sections were made through the venous outflow tract (therefore covering 1.5 -cm of outflow vein). Three sections were made through the graft-artery anastomosis at 1-mm intervals. These sections were mounted on gelatin-coated (or equivalent) glass slides and stained with hematoxylin and eosin or Verhoeff s elastin stain. [0137] The size of each of the tissue compartments, for example, the intima, the media and the lumen, were measured in microns. Each section was evaluated for the presence and/or extent of fibroblasts, fibrosis, abnormal collagen deposition, fibrin, graft fibrosis and graft infiltration. Scores were assigned for each variable on a scale of 0 through 4 (0 = no significant changes; 1 = minimal; 2 = mild; 3 = moderate; and 4 = severe). Representative grading criteria for histomorphologic findings are presented below in Table 1.

TABLE 1

HPF = High Powered Field

[0138] Additional sections of arteriovenous graft anastomotic sites from the 1- month animal test subjects only, were mounted on glass slides and stained with Verhoeff s elastin for morphometric analysis. Measurements of the luminal, medial, intimal and total vessel area were taken using computerized digital planimetry with a video microscope and customized software for each section. The extent of tissue remodeling was determined for each section by determining the intima area by the total vessel wall area (EEL) and by determining the lumen area. Venous luminal gain was measured angiographically by comparison of the venous lumen diameter post-surgery to that just prior to the one-month sacrifice. Luminal gain then was calculated by dividing each venous diameter by the reference graft diameter using the following equation: [late venous luminal gain] = [venous lumen diameter just prior to sacrifice / lumen graft diameter] - [venous lumen diameter post surgery / lumen graft diameter].

[0139] To examine the effects of the perivascular implants on matrix metalloproteinase (MMP) expression, venous tissue sections were subjected to immunohistochemical analysis. Five micrometer paraffin sections were cut and antigen retrieval performed by heating the sections for 20 minutes in high pH Target Retrieval Solution (Dako USA, Carpinteria, CA). The slides were covered with Peroxidase Block (Dako USA) for 5 minutes to quench endogenous peroxidase activity. Primary murine anti-human MMP-2 (1 :250 dilution, Chemicon International, Inc. Ternecula, CA) was applied for 45 minutes at room temperature and Primary rabbit anti-human MMP-9 (1 :250 dilution, Chemicon International, Inc. Ternecula, CA) was applied for 60 minutes at room temperature. All slides were counterstained with Mayer's hematoxylin (Sigma Chemical Co.). Porcine liver was used as a positive control and mouse IgGl or rabbit IgG were used as negative control. For every specimen, at least 6 non-overlapping fields were analyzed per section. For quantitative assessment of positive MMP staining, randomly selected areas were imaged using an Olympus BX60 microscope. Digital images (20Ox magnification) were captured and analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD). Each area of interest (e.g. intima, media and adventitia) was highlighted and positive staining was quantified by color segmentation. The results were expressed as percentage of positive stained area (positive area in mm² over total area in mm²).

[00102] A Pearson correlation was performed to determine the relationship between remodeling (i.e., lumen diameter) and MMP expression.

[0100] Results for Animal Subjects. Placement of the implantable material of the present invention at a site at or adjacent to a tubular or non-tubular tissue, structure or organ is effective to diminish fibrosis and negative tissue remodeling which follows a disruption of the tissue, for example, following a surgical intervention. For example, in the case of treated vascular tubular structures, the administration of the implantable material to the adventitia of such treated vascular tubular structures increases venous lumen (determined pathologically and angiographically) and vessel areas (determined pathologically) for an extended period of time following the initial intervention. Furthermore, the implantable material reduces MMP expression and/or activation, tissue fibrosis and abnormal collagen deposition of the tubular or non-tubular tissue, structure or organs. [0101] Subjects treated with the implantable material of the present invention as described above displayed one or more indicia of reduced negative tissue modeling, or positive tissue modeling. An indicia of positive remodeling is an increase in lumen diameter from baseline. The implants of the present invention increased lumen diameter by reducing negative tissue remodeling, fibroblast proliferation, abnormal collagen deposition and fibrosis. [0102] Lumen diameter was monitored using angiography of the arteriovenous graft anastomoses at the day of arteriovenous graft creation and just prior to 30-day sacrifice. Angiograms obtained at the day of creation and just prior to 1 -month sacrifice were evaluated both qualitatively and quantitatively in paired comparison to determine the extent of venous luminal gain. Pigs treated with the implantable material exhibited an increase in venous luminal gain at 1 -month of 1.8 ± 0.58 compared to venous luminal gain in pigs administered the control material of 0.01 ± 0.44, P < 0.05. The veins treated with the implantable material increased significantly in diameter from 1.04 ± 0.09 at day 0 to 2.9 ± 0.56 at 1 -month while veins administered the control material remained essentially unchanged from 1.28 ± 0.13 at day 0 to 1.29 ± 0.35 at 1 -month. Figure 5 is a graphical representation of the change in venous lumen diameter of subjects treated with the implantable material and subjects administered the control material at 3 days and one month. [0103] Morphometric analysis was also performed on pigs euthanized at 1 month. This data is represented below in Table 2 and agreed with the angiographic findings. Veins treated with the implantable material demonstrated a 2.3 fold increase in lumen area compared to grafts treated with the control material (P < 0.05). There was also a significant difference in total vessel area of the vein between the group treated with the implantable material and the control group, data presented below in Table 2, suggesting the implantable material has a positive effect on vessel remodeling. Total vessel area increased from 1 1.16 ± 3.4 mm² for control grafts to 20.72 ± 2.6 mm² for grafts treated with the implantable material (P < 0.05) suggesting positive remodeling in the grafts treated with the implantable material. At four weeks post-implant, there was no obvious difference in vessel remodeling observed at the arterial sites between groups (data not shown).

TABLE 2

[0104] Fibrosis was also present in the adventitia of veins in animals from both groups at 3 days and 1 -month, with greater fibrosis present at 1 -month consistent with the healing process. Fibrosis is characterized by an increase in the quantity and relative proportion of fibroblasts and of collagen material in a tissue sample. Fibrosis can be identified and quantified on hematoxylin and eosin stained sections as eosinophilic, hyalanized or fibrillar material. Less fibrosis was noted in the adventitia of veins treated with the implantable material compared to veins in control animals at 1 -month (an average difference of 1 severity point). TABLE 4

[0105] The implantable material of the present invention also reduced expression of matrix metalloproteinases in animals treated with the implantable material of the present invention. Immunohistochemical analysis of MMP-2 and MMP-9 positive cells in the total vessel, intima, media and adventitia 3-days and 1- month after surgery revealed reduced expression of MMPs in veins treated with the implantable material compared to veins administered the control material. [0106] In the control group, significant MMP-2 positive cells were observed in the adventitia, media and intima at day 3. MMP-2 positive cells were observed in tissue sections of animals administered the control material at a level of 1 1.2 ± 1.0% in the adventitia; 4.4 ± 0.6% in the media; and 2.1 ± 0.2% in the intima. In animals receiving the control material, positive staining for MMP-2 was predominantly located in the adventitia. At 1 -month, the amount of staining in animals administered the control material decreased in the adventitia (5.7 ± 0.9%) but remained increased in the media (4.9 ± 1.0%) and the intima (2.6 ± 0.8%). [0107] In the group treated with the implantable material, decreased expression of MMP-2 was observed in the adventitia, media and intima of vessels at day 3. MMP-2 positive cells were observed in tissue sections of animals treated with the implantable material at a rate of 6.9 ± 1.2% (P<0.05) in the adventitia; 2.3 ± 0.4% (PO.05) in the media; and 0.8 ± 0.2% (PO.05) in the intima. MMP-2 expression remained relatively unchanged from 3 days to one month in animals treated with the implantable material.

[0108] Figure 2 is a graphical representation of the expression of MMP-2 in stained tissue sections of subjects treated with the implantable material and subjects administered the control material at 3 days and at 1 month. A significant decrease in expression of MMP-2 in veins treated with the implantable material compared to veins administered the control material is evident. Decreased MMP-2 expression was observed in the intima, media and adventitia of veins treated with the implantable material at 3 days and at 1 month. [0109] MMP-9 expression was less intense at both time points for both animals receiving the control material and animals receiving the implantable material compared to MMP-2 expression, discussed above. At day 3, there was reduced staining in the adventitia of veins treated with the implantable material compared to control. At 1 month, decreased MMP-9 expression was observed in the intima and adventitia of the treated group compared to the control (P<0.05). Figure 3 is a graphical representation of the expression of MMP-9 in stained tissue sections of subjects treated with the implantable material and subjects administered the control material at 3 days and at 1 month. [0110] A Pearson correlation, presented in Figure 4, was performed for total MMP-2 expression and lumen diameter of tissue treated with the implantable material or a control material at one month. A strong correlation was observed between total MMP-2 expression and lumen diameter (r = -0.833, P < 0.05). [0111] Wishing not to be bound by theory, it is believed that the implantable material of the present invention restores the proteolytic balance, or the balance between MMPs and TIMPs, in structures treated with the implantable material. Tubular structures constitutively secrete MMPs and TIMPs in a very tightly controlled ratio. However, injury or disease of a tubular structure can induce a deviation in the MMP:TIMP ratio in the structure sufficient to initiate a cascade of events resulting in negative tissue modeling. The implantable material decreases expression of MMPs or increases expression of TIMPs to restore the balance between MMPs and TIMPs sufficient to restore positive tissue modeling to the treated structure. [0112] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims

ClaimsWhat is claimed is:

1. Use of an implantable material comprising endothelial or endothelial-like cells and a biocompatible matrix for the manufacture of an implant for inhibiting abnormal or pathological tissue remodeling.

2. Use of claim 1 wherein the abnormal or pathological tissue remodeling is inhibited at the site of or in the vicinity of or adjacent to the implant in vivo.

3. Use of claim 1 wherein the abnormal or pathological tissue remodeling is MMP-mediated.

4. Use of claim 1 wherein the inhibition of the abnormal or pathological tissue remodeling is TIMP-mediated.

5. Use of claim 4 wherein the inhibition of the abnormal or pathological tissue remodeling is mediated by one or more TIMP(s) secreted by the endothelial or endothelial-like cells of the implantable material.

6. Use of claim 1 wherein the abnormal or pathological tissue remodeling involves all or part of a tubular anatomical structure.

7. Use of claim 6 wherein the tubular anatomical structure is a vascular structure.

8. Use of claim 7 wherein the vascular structure is selected from the group consisting of a vein, an artery, an arteriovenous native fistula, an arteriovenous graft or a venous catheter.

9. Use of claim 1 wherein the abnormal or pathological tissue remodeling comprises cellular differentiation, infiltration, migration or proliferation.

10. Use of claim 9 wherein the cellular differentiation, infiltration, migration or proliferation is MMP-mediated.

1 1. Use of claim 1 wherein the abnormal or pathological tissue remodeling comprises extracellular matrix protein degradation.

12. Use of claim 1 1 wherein the extracellular matrix protein degradation is MMP-mediated.

13. Use of claim 1 wherein the abnormal or pathological tissue remodeling comprises fibroblast differentiation, infiltration, migration or proliferation.

14. Use of claim 13 wherein the fibroblasts are selected from the group consisting of adventitial fibroblasts, medial fibroblasts and myofibroblasts.

15. Use of claim 13 wherein the abnormal or pathological tissue remodeling comprises fibroblast migration to a luminal or interior portion of a tissue or organ.

16. Use of claim 1 wherein the abnormal or pathological tissue remodeling comprises fibrosis.

17. Use of claim 1 wherein the abnormal or pathological tissue remodeling comprises tissue thickening and/or adventitial fibrosis.

18. Use of claim 1 wherein the abnormal or pathological tissue remodeling causes luminal occlusion or narrowing of all or part of a tubular anatomical structure in vivo.

19. Use of claim 18 wherein the inhibition of abnormal or pathological tissue remodeling maintains the patency of all or part of the tubular anatomical structure in vivo.

20. Use of claim 1 wherein the implant is for adjunctive treatment with: (a) physical dilation of all or part of a tubular anatomical structure; and/or (b) surgical resection of fibrotic tissue; and/or (c) anti-matrix metalloproteinase drug treatment; and/or (d) anti-fibrotic drug treatment; and/or (e) anti- angiogenic drug treatment.

21. Use of claim 1 wherein the implant is for the treatment of a patient sub-group in which inhibition of MMP activity at the implant site is indicated.

22. Use of claim 1 wherein the implant is for the treatment of a patient sub-group in which physical dilation of all or part of a tubular anatomical structure precedes, follows or is coincident with implantation at the site of or in the vicinity of or adjacent to said physical dilation.

23. Use of claim 1 wherein the implant is for the treatment of a patient sub-group in which surgical resection of fibrotic tissue precedes, follows or is coincident with implantation at the site of or in the vicinity of or adjacent to said surgical resection.

24. Use of claim 1 wherein the implant is for the treatment of a patient sub-group in which adjunctive treatment precedes, follows or is coincident with implantation of the implant.

25. Use of claim 24 wherein the implant is for the treatment of a patient subgroup in which adjunctive MMP inhibitor treatment precedes, follows or is coincident with implantation of the implant.

26. Use of claim 24 wherein the implant is for the treatment of a patient subgroup in which adjunctive antifibrotic drug treatment precedes, follows or is coincident with implantation of the implant.

27. Use of claim 1 wherein said abnormal or pathological tissue remodeling is inhibited by locating the implant at, adjacent to or in the vicinity of said tissue remodeling in vivo.

28. Use of claim 27 wherein the implant is introduced at, adjacent to or in the vicinity of a tubular anatomical structure in vivo.

29. Use of claim 1 wherein the endothelial or endothelial-like cells secrete one or more tissue inhibitor metalloproteinase(s) (TIMP(s)) when implanted in vivo in association with said biocompatible matrix.

30. Use of claim 29 wherein the endothelial or endothelial-like cells secrete TIMP-2 when implanted in vivo in association with said biocompatible matrix.

31. Use of claim 1 wherein the endothelial or endothelial-like cells are selected from: a confluent population of cells; a near-confluent population of cells; a post-confluent population of cells; and cells which have a phenotype of any one of the foregoing populations of cells.

32. Use of claim 1 wherein the endothelial or endothelial-like cells are allogenic cells.

33. Use of claim 1 wherein the biocompatible matrix is a flexible planar material or flowable composition.

34. Use of claim 33 wherein the biocompatible matrix is a shape-retaining composition.

35. Use of an implantable material comprising endothelial or endothelial-like cells and a biocompatible matrix for the manufacture of an implant for treating an individual suffering from or susceptible to a condition which can be improved or prevented by inhibiting tissue remodeling.

36. Use of claim 35 wherein the tissue remodeling is abnormal or pathological tissue remodeling.

37. Use of an implantable material comprising endothelial or endothelial-like cells and a biocompatible matrix for the manufacture of an implant for treating an individual suffering from or susceptible to a condition which can be improved or prevented by inhibiting MMP activity.

38. Use of an implantable material comprising endothelial or endothelial-like cells and a biocompatible matrix for the manufacture of an implant for the treatment or prevention of adventitial fibrosis in a blood vessel.

39. A method of inhibiting abnormal tissue modeling, comprising the step of: providing to a recipient an implantable material comprising a biocompatible matrix; and anchored or embedded endothelial cells, endothelial-like cells, or analogs thereof, wherein said implantable material is provided to said recipient in an amount inhibit abnormal tissue modeling in said recipient.

40. An implantable material for inhibiting abnormal tissue modeling, wherein said implantable material comprises: a biocompatible matrix; and anchored or embedded endothelial cells, endothelial-like cells, or analogs thereof, wherein an effective amount of said implantable material inhibits abnormal tissue modeling in said recipient.

41. A method of reducing MMP expression and/or activation, comprising the step of: providing to a recipient an implantable material comprising a biocompatible matrix; and anchored or embedded endothelial cells, endothelial-like cells, or analogs of any of the foregoing; wherein said implantable material is provided to said recipient in an amount sufficient to reduce MMP expression and/or activation in said recipient.

42. An implantable material for reducing MMP expression and/or activation, wherein said implantable material comprises: a biocompatible matrix; and anchored or embedded endothelial cells, endothelial-like cells, or analogs of any of the foregoing; wherein an effective amount of said implantable material reduces MMP expression and/or activation in said recipient.

43. A method for the treatment or prevention of fibrosis, the method comprising the step of: providing to a recipient an implantable material comprising a biocompatible matrix; and anchored or embedded endothelial cells, endothelial-like cells, or analogs of any of the foregoing; wherein said implantable material is provided to said recipient in an amount sufficient to treat or prevent fibrosis in said recipient.

44. The method of claim 43 wherein the fibrosis is fibrosis of a vascular tissue, structure or organ.

45. The method of claim 43 wherein the fibrosis is adventitial fibrosis of a blood vessel.

46. A method for the treatment or prevention of luminal narrowing or occlusion of a tubular anatomical structure, the method comprising the step of: providing to a recipient an implantable material comprising a biocompatible matrix; and anchored or embedded endothelial cells, endothelial-like cells, or analogs of any of the foregoing; wherein said implantable material is provided to said recipient in an amount sufficient to treat or prevent luminal narrowing or occlusion in said recipient.

47. The method of claim 46 wherein the tubular anatomical structure is a structure of the vascular system system.

48. A composition suitable for use with the method of any one of claims 43 to 47.