CN101076360B - Materials and methods for minimally-invasive administration of a cell-containing flowable composition - Google Patents

Abstract

The disclosed invention is based on the discovery that a cell-based therapy can be used to treat, ameliorate, manage and/or reduce the progression of clinical sequelae associated with vascular interventions or cardiovascular diseases, particularly occlusive thrombosis, restenosis, intimal hyperplasia, inflammation and vasodilation. The invention further benefits from the additional discovery that a heretofore undescribed implantable flowable composition is capable of sustaining a confluent population of sufficiently viable cells which can be effectively administered via a minimally-invasive surgical procedure without diminishing the clinical effectiveness or the viability of the cells. The disclosed invention can be used to treat vasculature as well as non-vascular tubular structures such as a fallopian tube.

Description

But the materials and methods of the minimally-invasive administration of celliferous flow composition

The related application date

According to United States code 35 chapters and sections 119 (e), the non-temporary patent application of basis of December in 2005 submission on the 6th requires the temporary patent application U.S.S.N.60/634 of December in 2004 submission on the 8th, 155, the temporary patent application U.S.S.N.60/663 that on March 21st, 2005 submitted to, 859, the temporary patent application U.S.S.N.60/682 that on May 19th, 2005 submitted to, 054, and the priority of temporary patent application U.S.S.N.60/; The full content of above-mentioned each patent application all is incorporated herein by reference.

Background of invention

Cardiovascular disease causes in the whole world over 500,000,000 people's death every year, in the U.S., causes 1 million people dead., in the U.S., approximately complete 1,500,000 operations every year to attempt to alleviate the obstructive arterial disease that causes because of these idiogenesis.Several balloon angioplasty and laser angioplasty, speckle rotary-cut art, stent placement and bypass graft etc. of comprising of these operations that can mention.Angioplasty, blood vessel by-pass grafting even the long-term effect of transplant operation owing to having promoted the generation of arteriopathy to be restricted after these programs.The disappearance of interior film integrality, occluding thrombus disease and to cause smooth muscle cell spasm, migration and the propagation of neointimal hyperplasia be the Typical Representative of this class arteriopathy.For example, restenosis causes the 20-50% in these patients that the obstructive arterial disease occurs.In 3-6 after coronary angioplasty month, surpass 1/3rd extra interventional procedure, the even by-pass operation of another time angioplasty of needs of patients.The speckle Atherectomy devices is not good yet, and along with patient's number of accepting this operation increases, the ratio that restenosis occurs is soaring to 47% from 10%.The use of endovascular stent is same in this, and some is disappointing.A recent research report, in initial several days after support inserts, acute complications occurs in the patient except about 5%, and outside closing suddenly, the ratio that restenosis occurs is 35%.

Observed similar problem in accepting the patient of vascular bypass operation.The average life that saphena aorta-coronary artery inserts transplantation is only 7 years.Have 10% inaccessible in postoperative first few weeks in all these class grafts.During 1 year and 5 years, 20% and 30% graft generation obturation is arranged respectively.Be subjected to the arteriovenous fistula of the dialysis patient of same pathology domination to limit the effect of hemodialysis.

The arteriopathy that promotes such as the characteristics of restenosis are the depositions of a large amount of smooth muscle cell proliferation and a large amount of extracellular matrixs of being produced by these cells.What become apparent at present is, has a common initial pathology event between the arteriopathy of promotion from body atherosclerosis and mechanicalness interventional procedure after, that is, and and the disappearance of endothelial cell integrity and function.

Inner hypophloeodal single-layer is arranged along arterial wall, and as the physiological dual bioregulator of blood vessel, works.Endothelium is continuous by forming between blood circulation and arterial wall, the permeable non-thrombinogen barrier of selectivity, and the integrity of structure is provided for blood vessel.Yet, what recognize gradually is that endothelium also produces and supply with the product that can control blood flow, blood vessel degree of tightness state (vessel tone), occluding thrombus disease, platelet activation, adhesion and gathering, leukocyte adhesion, mononuclear cell infiltration and smooth muscle cell migration and propagation., because the smooth muscle cell mitogen is prevalent in arterial wall, be that the existence of complete endothelium makes normal blood vessels maintain resting state.In vessel endothelium and induced endothelial or after removing, the compound of being secreted by endothelium is removed similarly, and starts to occur sequence of events, thus the obstructive arterial disease that has caused smooth muscle cell proliferation out of control and migration to cause.

The many clinical interventional procedure that is used for the treatment of at present cardiovascular disease, comprise coronary angioplasty, intracoronary stening and speckle rotary-cut art, can adopt Noninvasive closed operation method to complete.These Noninvasive intravascular interventional therapeutic procedure strategies should be got involved position to treat the intravascular modalities of therefore impaired or ill target endothelium with the delivery of therapeutic material that has equally minimally-invasive to blood vessel.

In addition, present method to endovascular delivery therapeutic material depends on the surface of internal cavity that material is imposed on blood vessel.Yet,, owing to contacting of blood circulation, having limited active effect and persistent period, to surface of internal cavity, bestow the therapeutic material or reagent only can provide temporary transient effect to impaired or ill target endothelium.

An object of the present invention is to provide to being positioned at impaired or ill inner chamber endothelium position, or adjacent with this position, or the materials and methods of the generation of occluding thrombus disease, restenosis, neointimal hyperplasia and other clinical sequela relevant to blood vessel intervention or cardiovascular disease non-invasively or minimally-invasive ground delivery of cells therapeutic preparation, and is reduced in position thereupon outside near the chamber this position or around blood vessel.

Summary of the invention

The present invention has utilized such discovery: but transplant among implantable flow composition, on or within cell can be designed for multiple minimally-invasive delivery modality, for example, at tubular anatomical structures chamber outer surface, or in the adjacent position of this outer surface, or carry out depositing around intravascular administration and blood vessel near this outer surface, described anatomical structure is such as but not limited to blood vessel.At the outer surface of tubular anatomical structures, or on outer surface, or the minimally-invasive of external surface peripheral is sent and is also included within the present invention.In the situation that blood vessel, materials and methods of the present invention is suitable for treating the clinical sequela relevant to blood vessel intervention or cardiovascular disease with control.

On the one hand, but the present invention is the flow composition of the impaired or disease sites for the treatment of tubular anatomical structures inner chamber, but flow composition wherein comprises biocompatible matrix and cell, but and the amount of flow composition wherein be the amount that can effectively treat impaired or disease sites.According to an embodiment, tubular anatomical structures is blood vessel.According to some embodiments, but flow composition is so that such as smooth muscle cell proliferation, occluding thrombus disease, neointimal hyperplasia, restenosis, amount acute or chronic inflammatory disease or vasodilation etc., providing of impaired or disease sites effectively to be provided.For purpose of the present invention, but refer to can be with the compositions of injecting or the injection type delivery apparatus is bestowed such as but not limited to pin, syringe or conduit for flow composition.Other delivery apparatus that adopts extruding, sprays or discharge is also included within the present invention.

According to an embodiment, but the cell of flow composition is endotheliocyte or cell with endotheliocyte sample phenotype.According to another embodiment, described cell is the coculture of two or more cell type.This two or more cell type is selected from endotheliocyte, epithelial cell, smooth muscle cell, fibroblast, stem cell, endothelial progenitor cells and myocardial cell.Being applicable to cell product of the present invention can be available from single donor or a plurality of donor.

According to another embodiment, biocompatible matrix is gel, foam or suspension.In another embodiment, biocompatible matrix comprises microgranule or microcarrier.In certain embodiments, microgranule or microcarrier further comprise gelatin, collagen protein, fibronectin, fibrin, laminin，LN or attaching peptide.An exemplary attaching peptide is the peptide of sequence arginine-Gly-Asp (RGD).According to another embodiment, the diameter of microgranule or microcarrier is approximately 20 microns to approximately 500 microns, and preferred diameter is approximately 200 microns.

In another embodiment, but flow composition further comprises carrier fluid.In especially preferred embodiment, remain unchanged but flow composition is shape, thereby allow the practitioner to control to the deposition at the particular deposition position of appointment on the degree of necessity.

On the other hand, the present invention is the method for the impaired or disease sites for the treatment of tubular anatomical structures inner chamber.The method comprises makes the impaired or disease sites that is positioned at the tubular anatomical structures inner chamber, or adjacent with this position, but near or the step that contacts with flow composition of the chamber outer surface of the tubular anatomical structures this position.Surface, non-chamber (outside chamber) herein can be outer surface or the circumvascular surface of blood vessel.For purpose of the present invention, outside non-chamber or chamber, position is any position except the inner surface in chamber.In the situation that blood vessel, for example, outside chamber or exocoel position can be in the adventitia of blood vessel, middle film or inner membrance; In the situation that non-blood vessel tubular anatomical structures, position, corresponding non-chamber all within the scope of the invention.

According to an embodiment,, by passing or see through the inwall of tubular anatomical structures, then be positioned at impaired or disease sites, or adjacent with this position, but or near the outside deposition flow composition of the tubular anatomical structures this position complete and send., according to another embodiment, but further being included in the chamber outer surface of tubular anatomical structures, this method identifies the step at the position of deposition flow composition.According to an embodiment, this authentication step occurs in this and passes or, through before step, perhaps with this, pass or see through step and occur simultaneously.In one embodiment, this authentication step is completed by imaging.In another embodiment, this authentication step is completed by palpation.

In one embodiment, by through percutaneous dosing intravasation week gap, then at impaired or disease sites, or adjacent with this position, but or near the outer surface of the tubular anatomical structures this position the deposition flow composition complete and send., according to another embodiment, but further being included in the outer surface of tubular anatomical structures, the method identifies the step at the position of deposition flow composition.This authentication step can occur in before this enters step, perhaps with this, enters step and occurs simultaneously.According to an embodiment, this authentication step is completed by imaging.In another embodiment, this authentication step is completed by palpation.

According to the various embodiments of this method, the outer surface of tubular anatomical structures is surface, non-chamber or occupies at the described perivascular canal in this paper other places.According to a preferred embodiment, tubular anatomical structures is blood vessel.According to another preferred embodiment, blood vessel comprises support.In a further preferred embodiment, the tubular anatomical structures that is treated is non-blood vessel structure, such as but not limited to fallopian tube.

The accompanying drawing summary

Fig. 1 is the typical cells growth curve according to an illustrative embodiment of the present invention.

Detailed Description Of The Invention

As this paper explain, the present invention is based on such discovery: the therapy based on cell can be used for the treatment of, alleviation, control and/or minimizing and blood vessel is got involved or cardiovascular disease is relevant clinical sequela, especially occluding thrombus disease, restenosis, neointimal hyperplasia, inflammation and vasodilative progress.The present invention further benefits from another discovery, namely, but unrecorded flow composition still up to now, microparticle formulation for example, the cell mass of the vigor abundance that is paved with can be provided, said composition comprise transplant in biocompatible matrix for example among implantable microparticle material, on or within cell, and said composition can adopt for example endovascular delivery or the effective administration of local dermal delivery in the closure program of minimally-invasive administration pattern, but and does not reduce the survival rate of the transplanted cells of clinical effectiveness or implantable flow composition.The teaching of below listing is to manufacturing and use materials and methods of the present invention that sufficient guidance is provided, and further to standard and the experimenter who determines the suitable performance that is used for test, measurement and monitoring materials and methods of the present invention, provides sufficient guidance.

Therefore, developed for the therapy based on cell of controlling clinically blood vessel intervention or cardiovascular disease.Exemplary embodiment of the present invention comprises biocompatible matrix and the cell that is applicable to treatment example described herein.Especially, in a preferred embodiment, but implantable flow composition comprises biocompatible matrix and endotheliocyte or endothelioid cells.In a further preferred embodiment, but implantable flow composition comprises endotheliocyte or endothelioid cells, preferred human aorta endothelial cell and particle type biocompatible matrix.

But implantable flow composition of the present invention comprise transplant on biocompatible matrix, among and/or within cell.The meaning of transplanting refers to through cell, cell and/or cell be adhered to securely to the interaction of substrate, so that cell can sustain the harsh conditions of preparation operation disclosed herein.As explain in this paper other places, but an effective embodiment of implantable flow composition comprises having closely being paved with cell mass, being paved with cell mass or being paved with rear cell mass of preferred phenotype.Natural is, in preparation property operating period, but probably exfoliative cyte and/or some cell do not resemble other cell and adhere to securely the embodiment of implantable flow composition.All essential conditions are, but the cell that implantable flow composition comprises should meet the standard of function described herein or phenotype.

But implantable flow composition of the present invention is developed according to the tissue engineering principle, and has represented the new method that meets above-mentioned clinical demand.But the unique distinction of implantable flow composition of the present invention is, transplant on biocompatible matrix, among and/or within living cells can provide to tubular anatomical structures the product based on various kinds of cell that is in the physiology ratio under the physiology feedback control.As described elsewhere herein, but the cell that is applicable to implantable flow composition is endotheliocyte or endothelioid cells.The local delivery of the multiple compounds that is undertaken by these cells and physiological dynamics administration provide more effective adjusting to the process of being responsible for keeping functional intracavity skin.Importantly, but for example the endotheliocyte in implantable flow composition of the present invention has been avoided the destruction of the aggressivity blood flow of intravascular space because it preferably is placed in position outside non-chamber or chamber.

But with parcel, deposition or alternate manner and be positioned at impaired or ill target chamber, or adjacent with this target chamber when implantable flow composition of the present invention, or during the contact of outside near the chamber this target chamber or position, non-chamber, it is used for rebuilding homeostasis.Namely when the external administration of chamber, but implantable flow composition of the present invention can provide, simulate supportive physiological environment, and help lend some impetus to the growth of functional inner chamber.As this paper expection, tubular anatomical structures is the structure with surface of internal cavity and chamber outer surface.In some structure, surface of internal cavity is endothelial layer; In the other structure, surface of internal cavity is non-endothelial layer.In addition, for purpose of the present invention, as described in this paper other places, outside chamber or surface, non-chamber can be but be not limited to is the outer surface of tubular structure.

For example, endotheliocyte can discharge plurality of reagents, these reagent join together to suppress or alleviation gets involved to blood vessel or cardiovascular disease after the relevant bad physiological event of acute complications.As this paper was illustrative, the compositions of recurrence normal physiological and using method and administration can strengthen the functional of endothelium, and improve the long-term patency rates of this intracavity skin.Normally, treatment is included in impaired or ill target endothelium place, or in this target endothelium adjacent position, or near this target endothelium, for example, but at the perivascular canal of outside, target blood structure chamber, deposit implantable flow composition of the present invention.When deposition or otherwise contact impaired, be wound or during diseased vessel, but the cell of implantable flow composition can be to the target blood structure, for example endovascular bottom smooth muscle cell improves the growth regulating compound., although in the blood vessel outside, but implantable flow composition of the present invention provides the effective supply of the multiple modulating compound that comes from cell, but avoided producing the mechanism of the blood flow in intravascular space simultaneously.

Impaired or diseased vessel can promote normal or near normal the recovery and normal physiological with the preferred embodiments of the invention treatments.By contrast, in the situation that lack with the preferred embodiments of the invention treatment, the recovery of normal physiological is damaged, for example, after blood vessel intervention or cardiovascular disease, can be to be exceedingly fast or velocity anomaly growth out of control from body endotheliocyte and smooth muscle cell.Therefore, as this paper expection, but the treatment of employing implantable flow composition of the present invention will produce the normal or near normal recovery of the autologous tissue at blood vessel intervention or cardiovascular disease position, for example, be enough to keep normal or near normal blood vessels patency rate.

But implantable flow composition of the present invention can be placed in and be treated blood vessel structure with multiple configuration.Blood vessel can all or part ofly contact; For example, but implantable flow composition of the present invention can along the circumferential direction or with arc be applied to blood vessel.But blood vessel only needs to contact with a certain amount of functional implantable flow composition that is enough to improve blood vessel structure.

For purpose of the present invention, contact refer to directly or indirectly as with defined chamber, this paper other places outside or non-chamber surface interaction.In the situation that some preferred embodiment, actual physical contact is not that effect is necessary.In other embodiments, actual physical contact is preferred.All implement condition essential to the invention, at impaired or disease sites, or in the adjacent position at this position, or outside near the chamber this position or position, non-chamber deposition can effectively be treated the implantable material of the amount of impaired or disease sites.In the situation that some i or I, ill or damaged part can find expression in surface of internal cavity clinically.In the situation that Other diseases or damage, ill or damaged part can find expression in surface, outside chamber or non-chamber clinically.In some diseases or damage, ill or damaged part can find expression in surface, outside surface of internal cavity and chamber or non-chamber clinically.The present invention can effectively treat any aforementioned clinical manifestation.

But the embodiment of implantable flow composition of the present invention can be applied to any interventional therapy that needs to keep the tubular anatomical structures of homeostasis.As this paper expection, tubular anatomical structures is to have outside surface of internal cavity and chamber or the structure on surface, non-chamber.For purpose of the present invention, the chamber outer surface can be but be not limited to the outer surface of tubular structure.In some tubular structure, surface of internal cavity is endothelial layer; In some other structure, surface of internal cavity is non-endothelial layer.The present invention can effectively treat the tubular structure that is lined with endotheliocyte or is lined with non-endotheliocyte.

Tubular anatomical structures comprises the structure of the ventricular system of vascular system, reproductive system, urinary system, gastrointestinal system, lung system, respiratory system and brain and spinal cord.The representative example of tubular anatomical structures comprises passage, vagina vasorum and the brain in passage, deferent duct and other male genetic road of tremulous pulse and vein, tear stains, trachea, bronchus, bronchioles, nasal meatus (comprising nasal sinuses) and other air flue, pharyngotympanic tube, external auditory meatus, oral cavity, esophagus, Stomach duodenum, small intestinal, large intestine, biliary tract, ureter, bladder, urethra, fallopian tube, uterus, vagina and other female genital tract and the ventricular system (cerebrospinal fluid) of spinal cord.For purpose of the present invention, tubular anatomical structures can be natural or non-natural, such as but not limited to the anastomotic stoma of operation generation.

Impaired or ill endothelium: in some preferred embodiment, can get involved and include but not limited to that angioplasty, speckle rotary-cut art, intravascular stent (comprising bare mental stents and bracket for eluting medicament), vascular bypass operation (comprising artery bypass grafting art and peripheral arterial bypass graft), organ transplantation, arteriovenous fistula and other vascular anastomosis form art, arteriovenous, periphery and other is migrated to the shape art with the blood vessel that causes blood vessel injury of the present invention treatment, and the angioaccess associated injury that occurs subsequently, comprise the pricking wound or other interventional therapy that obtain between the intravasation dialysis period.Every kind of intervention all produces to a certain degree damage to the endotheliocyte lining of lumen of vessels.Otherwise, damaged blood vessels chamber experience cascade biochemical event, thereby cause the appraisable sequela of various clinical, include but not limited to the formation of occluding thrombus disease, restenosis, neointimal hyperplasia, acute and chronic inflammation, smooth muscle cell proliferation, vascular remodeling, vasodilation and vulnerable plaque pathological changes.

Thrombosis or occluding thrombus disease are relevant with hematoblastic adhesion, gathering and machineization; Occluding thrombus disease is usually relevant to organized thrombus.Thrombosis is characterised in that the blood flow disappearance in thrombus area.The antithrombotic compound that endothelium or endothelioid cells discharge includes, but are not limited to Heparan sulfate proteoglycan, prostacyclin and nitric oxide.But with implantable flow composition treatment of the present invention, can improve the patency rate that is treated blood vessel.

Narrow, restenosis, neointimal hyperplasia are characterised in that and the obstructive pulmonary disease of smooth muscle cell in the relevant lumen of vessels of intracavity vigorous growth with smooth muscle cell proliferation.Endothelium or endothelioid cells discharge the compound that suppresses smooth muscle cell proliferation in cavity region.The exemplary therapeutic compound that is produced by endothelium or endothelioid cells includes, but are not limited to Heparan sulfate, TGF-β and nitric oxide.Narrow, restenosis, neointimal hyperplasia and smooth muscle cell proliferation are identified by for example angiography, intravascular ultrasound or other ultrasonic technique.But with implantable flow composition treatment of the present invention, can reduce narrow percentage rate, inaccessible degree and/or improve the patency rate relevant to being treated blood vessel

The raising, adhere to and permeate relevantly of inflammation and inflammatory cell, inflammatory cell includes but not limited to granulocyte, neutrophilic granulocyte, mononuclear cell, macrophage and lymphocyte.In addition, the increase of vascular permeability causes body fluid, immunoglobulin, complement and the local accumulation of other blood protein in the adjacent tissue of damage location, the expression that it is induced conversely with the adhesion molecule of the surface combination of circulating monocytic cell and neutrophilic granulocyte, increase the speed that the phagocyte migration is passed the surface, chamber and entered adjacent tissue greatly.After activation, these cells can discharge hydrolytic enzyme, cytokine, chemotactic factor and somatomedin.In the chronic progressive stage of inflammation, damaged part becomes has fibrous cap to be coated with, and this fibrous cap covers on fat core and slough.Endothelium or endothelioid cells discharge the anti-inflammatory compound that can reduce inflammatory reaction in cavity region.But with implantable flow composition treatment of the present invention can the inflammation-inhibiting cell active and/or gather, therefore generation and the secretion of somatomedin have been reduced, and reduced the local vascular infiltration of macrophage, thereby the acute inflammatory reaction of prevention, minimizing or alleviating vascular damage location.The alleviation of acute inflammatory reaction or prevention can interrupt causing the event of chronic inflammatory disease, thereby final chamber infringement and/or dysfunction of blood vessel are minimized.In addition, the alleviation of chronic inflammation tissue or rehabilitation can be lowered for example risk of angiopathy (including but not limited to vulnerable plaque or atherosclerosis) outbreak of long-term risk, and described angiopathy includes but not limited to vulnerable plaque and atherosclerosis.

In addition, can include but not limited to the spontaneous cardiovascular disease of the present invention treatment as the vulnerable plaque pathological changes of acute and chronic inflammation, occluding thrombus disease, neointimal hyperplasia, restenosis, smooth muscle cell proliferation, vasodilation, negativity vascular remodeling, blood vessel structure intracavity and various unstability superior mesenteric artery syndromes etc.Other can comprise any ischemia, hypoxgia or faulted condition of comparing the blood supply quantity not sufficient with demand with the rapid wear vascular disorder of the present invention's treatment.The rapid wear vascular disorder can be produced by damage or the reparation of any negative effect blood supply.Exemplary rapid wear disease comprises the unstability superior mesenteric artery syndrome, such as ischemia, unstable angina pectoris (comprise scope from exercise induced angina pectoris to the tranquillization type anginal unstability scope), aorta ischemia, periphery ischemia (comprising the disease scope of scope from the intermittent claudication to the gangrene), intestinal ischemia and renal ischaemia etc.

In certain embodiments of the invention, at blood vessel for the first time, get involved, but for example adopt the impaired or ill target endothelium of implantable flow composition treatment of the present invention when angioplasty, stent endoprosthesis or anastomosis.This treatment can reduce to get involved by blood vessel the damage cause, and for example the endothelium that causes of angioplasty is exposed.According to other embodiment, but implantable flow composition be applied to the rescue blood vessel after getting involved impaired or ill target endothelium and to get involved relevant clinical arteriopathy, include but not limited to the development of restenosis for example or occluding thrombus disease.

In certain embodiments of the invention, but before giving implantable flow composition, simultaneously and/or given afterwards extra therapeutic agent.For example, can prevent or reduce the reagent of blood clotting formation, platelet aggregation or other similar obturator.Exemplary reagent comprises, for example Heparan sulfate and TGF-β.According to the indication of implanting, but can also add other cytokine or somatomedin in implantable flow composition, comprise the VEGF that promotes again endothelialization and promote the complete b-FGF of blood vessel.The therapeutic agent of other type includes but not limited to antiproliferative and antitumor agent.Example comprises rapamycin, paclitaxel and E2F Decoy reagent.Any aforementioned agents all can part or whole body administration; If be topical, but some reagent can be included in implantable flow composition.

Cell derived: such as described herein, but implantable flow composition of the present invention comprises cell.Cell can be allochthonous, xenogenesis or from body.In certain embodiments, the source of living cells can derive from suitable donor or a plurality of donor.In some other embodiment, the source of cell can derive from corpse or cell bank.

In an at present preferred embodiment, cell is endotheliocyte.In an especially preferred embodiment, these endotheliocytes are available from vascular tissue, and are preferred but be not limited to arterial tissue.As hereinafter illustrative, a kind of applicable vascular endothelial cell type is aortic endothelial cell.Another kind of applicable vascular endothelial cell type is huve cell.Another applicable vascular endothelial cell type is coronary artery endothelial cell.Other is applicable to vascular endothelial cell type of the present invention and comprises pulmonary artery endothelial cell and iliac endothelial cells.

In another at present preferred embodiment, suitable endotheliocyte can be available from non-vascular tissue.Non-vascular tissue can derive from the described tubular anatomical structures in any this paper other places, perhaps can derive from any non-vascular tissue or organ.

In another embodiment, endotheliocyte can derive from endothelial progenitor cells or stem cell; In another embodiment, endotheliocyte can derive from CFU-GM or stem cell usually.In other preferred embodiment, cell can be the allogeneic that derives from blood vessel or non-vascular tissue or organ, xenogenesis or from the non-endotheliocyte of body.The present invention also comprises aforementioned any cell of making through genetic modification, genetic modification or genetic engineering.

In further embodiment, with the co-culture of cells of two or more type, to prepare compositions of the present invention.For example, the first cell is imported in the implantable material of biocompatibility, and be cultured to and be paved with.The first cell type comprises, for example combination of the cell type of the cell type that is suitable for producing the environment that helps endothelial cell growth of smooth muscle cell, endotheliocyte, fibroblast, stem cell, endothelial progenitor cells, myocardial cell, smooth muscle cell and fibroblastic combination, other any needs or needs.In case the first cell type reaches the state of being paved with, the second cell type is seeded in be positioned among the implantable material of biocompatibility, on or within the first be paved with on cell type, and be cultured to the first cell type and the second cell type all reaches the state of being paved with.The second cell type comprises, for example combination of the cell type of endotheliocyte or other any needs or cell type.First and second kinds of cell types may comprise the identical cell type that derives from two or more different donors or source.First and second kinds of cell types can progressively import, and perhaps as single mixture, import.Also can change the density of cell, thereby for AV, transplant the ratio of smooth muscle cell Human Umbilical Vein Endothelial Cells is become approximately 2: 1, for the periphery coronary artery bypass grafting, ratio be become approximately 1: 1, perhaps other is fit to the ratio of other clinical practice.

, for prevention has the smooth muscle cell of hyper-proliferative tendency or the hyper-proliferative of other cell type, can revise the cultivation program.For example, after the first cell type is paved with, before importing the second cell type, can first culture be covered with the attachment element that is suitable for the second cell type.Exemplary attachment element comprises with gelatin and covers culture, to improve the tack of endotheliocyte., according to another embodiment, between the second cell type culture period, add heparin can reduce the propagation of the first cell type in culture medium, and optimize the first cell type of needs and the ratio of the second cell type.For example, after the smooth muscle cell initial growth, can give heparin to control the growth of smooth muscle cell, thereby reach the larger endotheliocyte ratio to smooth muscle cell.

In a preferred embodiment, at first by smooth muscle cells inoculation to biocompatible matrix is produced coculture to produce blood vessel structure., in case smooth muscle cell reaches the state of being paved with, endotheliocyte is inoculated on the smooth muscle cell of cultivating on implantable material, to produce simulated blood vessel.This embodiment can be applied to for example AV according to methods described herein transplants or the periphery bypass graft, to promote the integration of prosthese graft materials.

The necessary condition of the cell of all compositionss of the present invention is to show one or more preferred phenotype or functional characteristics.As this paper is previously described, the present invention is based on such discovery: the cell with easy evaluation phenotype is when with preferred biocompatible matrix (in this paper other places, describing) associating, and it can promote, repair and/or otherwise regulate and treat the impaired of impaired or ill target vessel endothelium or another tubular anatomical structures or endotheliocyte physiology and/or intracavity ambient stable that ill target chamber is relevant.

For purpose of the present invention, a kind of preferred typical cells phenotype of easily identifying of the present invention is by hereinafter described analyzed in vitro method measurement, can suppress or otherwise disturb the phenotype of smooth muscle cell proliferation.This paper is referred to as the inhibition phenotype.

Other that is shown by the cell of compositions of the present invention identifies that easily phenotype is that antithrombotic maybe can suppress the phenotype of platelet adhesion and gathering.Antithrombotic acitivity can be used hereinafter described external Heparan sulfate analytic process and/or extracorporeal platelet aggregation assay.

In the effective embodiment of typical case of the present invention, the phenotype that cell need to show is no more than a kind of in aforementioned phenotype.In certain embodiments, cell can show more than one aforementioned phenotypes.

Although aforementioned phenotype represents a kind of functional endotheliocyte separately,, such as but not limited to vascular endothelial cell, show that the non-endotheliocyte of this (these) phenotype is considered from purpose of the present invention, think endothelioid cells, therefore be applicable to the present invention.This paper also is called endothelioid cells the functional analog of endotheliocyte, or the functional analogies of endotheliocyte.Therefore, only for illustrating purpose, the cell that is applicable to materials and methods disclosed herein also comprises stem cell or the CFU-GM that produces endothelioid cells; Origin is for non-endotheliocyte but be similar to cell through the endotheliocyte of parameter decision described herein in function performance; Any origin through engineered or otherwise modify after, have the functional cell of endothelium sample through parameter decision described herein.

Normally, cell mass after being paved with when being present in, closely being paved with or being paved with, and with preferred biocompatible matrix those whens associating as described herein, cell of the present invention demonstrates one or more above-mentioned phenotypes.It will be recognized by one of ordinary skill in the art that, being paved with, closely being paved with or being paved with rear cell mass can easily identify by multiple technologies, and wherein the most frequently used and technology that extensively generally acknowledged is direct microexamination.Other technology comprises and adopts the standard cell lines counting technology, assesses the cell number of per unit surface area such as but not limited to hematimeter or Coulter-counter.

In addition, for purpose of the present invention, when endothelioid cells includes but not limited to parameter measurement described herein, imitate on function and phenotype or simulation closely be paved with, be paved with and be paved with after the cell of endotheliocyte.

Therefore, utilize and hereinafter described describe in detail and instruct, but those of ordinary skills will understand effective embodiment of how making, use, test and identifying implantable flow composition disclosed herein.That is exactly, but teaching disclosed herein provides all manufacturings and used the necessary condition of implantable flow composition of the present invention.In addition, teaching disclosed herein provides all effectively to identify, make and use the necessary condition of the compositions useful that is equal to cell that contains.Finally, all essential conditions are, according to these equivalents of method disclosed herein, can effectively treat, control, regulate or alleviate chamber damage or disease, for example as non-limitative example, to chamber that blood vessel is got involved or cardiovascular disease is relevant, damage or disease.As skilled practitioner will understand, being equal to embodiment and can only adopting normal experiment and teaching provided herein to identify of compositions of the present invention.

In some preferred embodiment, but be used for the aorta of the endotheliocyte separation of implantable flow composition of the present invention from people's cadaveric donors.Every batch of cell all can derive from single or multiple donors.The existence of every batch of its endotheliocyte purity of the equal extensive testing of cell, biological function, antibacterial, fungus, known person pathogen and other external reagent.Cell, with the freezing preservation of known technology and stock, is cultivated expansion after treating, make subsequently implantable compositions.

The cell preparation: as mentioned above, suitable cell can be available from various types of organizations and cell type.In some preferred embodiment, but be used for the aorta of the human aorta endothelial cell separation of implantable flow composition from cadaveric donors.In other embodiments, Porcine aorta endothelial cells (Cell Applications, San Diego, CA) by with separate the similar program of human aorta endothelial cell and separate from the normal pig aorta.Every batch of cell all can derive from single or multiple donors, then the existence of its endotheliocyte survival rate of extensive testing, purity, biological function, mycoplasma, antibacterial, fungus, yeast, known person pathogen and other external reagent.With known technology with cell further cultivate, evaluation and freezing preservation,, to form the working cell storehouse in the 3rd to the 6th generation, cultivate expansion after treating, make subsequently the implantable compositions of biocompatibility.

People or Porcine aorta endothelial cells add the endothelial cell growth medium approximately to prepare in the pretreated T-75 bottle of 15ml at every bottle.Human aorta endothelial cell is preparation in endothelial cell growth culture medium (Endothelial Growth Media, EGM-2, Cambrex Biosciences company, EastRutherford, New Jersey).EGM-2 is comprised of endothelial basal medium (Endothelial Basal Media, EBM-2, Cambrex Biosciences company) and the EGM-2 SingleQuots that contains 2%FBS.Pig cell prepares in the EBM-2 that is added with 5%FBS and 50 μ g/ml gentamycins.Culture bottle is placed in couveuse, at approximately 37 ℃, 5%CO ₂Kept minimum 30 minutes under the condition of/95% air and humidity 90%.One bottle or two bottles of cells are taken out refrigerator from-160 ℃ to-140 ℃, approximately thawing under 37 ℃.Every bottle of cell after thawing is with preferred approximately 3 * 10 ³Individual cell/cm ³, but be not less than 1.0 * 10 ³Individual/cm ³And be not more than 7.0 * 10 ³Individual/cm ³Density be seeded in two T-75 bottles; The culture bottle that then will contain cell is put back in couveuse.Approximately after 8-24 hour, remove culture medium (spent media), and replace with fresh culture.After this, change a subculture in every 2-3 days, until cell reaches preferred approximately 85-100%, but be not less than 60% and be not more than 100% degree of being paved with.When but implantable flow composition is intended for use when clinical, but only the antibiotic-free culture medium can be used for the production of thaw rear cultivation and the implantable flow composition of the present invention of human aorta endothelial cell.

Then remove the endothelial cell growth culture medium, cell monolayer rinses with 10ml HEPES buffer saline (HEPES).Remove HEPES, add the 2ml insulin so that cell breaks away from from T-75 bottle wall.After breaking away from generation, add 3ml insulin neutralization solution (TNS) to stop enzyme reaction.Add in addition 5ml HEPES, cell is counted with hematimeter.Cell suspending liquid is centrifugal, in the situation that people's cell, with antibiotic-free EGM-2 regulating density to approximately 1.75 * 10 ⁶Individual cell/ml, or in the situation that pig cell, with the EBM-2 regulating density that is added with 5%FBS and 50 μ g/ml gentamycins to approximately 1.50 * 10 ⁶Individual cell/ml.

Biocompatible matrix: according to the present invention, but but a preferred embodiment of implantable flow composition comprises form is the biocompatible matrix of gel, foam, suspension, microcarrier, microcapsule flow fiber structure or other flowable materials.Biocompatible matrix allow Growth of Cells and be attached to substrate, thereon or in substrate.When implanting the outer surface of blood vessel for example, biocompatible matrix can rest on approximately 7-90 days of implant site before it is by bioerosion, preferably at least about 7-14 days, more preferably at least about 14-28 days, most preferably at least about 28-90 days.

For purpose of the present invention, but flow composition refer to can with inject or the injection type delivery apparatus such as but not limited to the compositions of pin, syringe or catheter drug delivery.Other delivery apparatus that adopts extruding, sprays or discharge is also included within the present invention.The biocompatible matrix of any non-solid form for the injection type delivery apparatus includes in the present invention, and device wherein can be implemented intravascular administration or local percutaneous dosing by along internal blood vessel length, advancing.But preferred flow composition is that shape remains unchanged.As this paper expection; but the implantable flow composition that contains the cell that is implanted into the matrix of microparticles that can flow; be designed to any injection delivery apparatus, this device comprises that inside diameter ranges is No. 22 standard size to 26 standard sizes, and length range is about 1 to 20mm pin.Preferred injection delivery apparatus transmissibility approximately 50mg, approximately 1 to the implantable microparticle material that contains 100 ten thousand cells of having an appointment in about 3ml medium.

According to general preferred embodiment of the present invention, but flow composition comprises the biocompatibility matrix of microparticles, for example derives from the product G elfoam of pig skin gelatinum

Microgranule, Gelfoam Powder or powdery Gelfoam

(Pfizer Inc. company, New York, New York) (hereinafter referred to as " Gelfoam microgranule ").According to another embodiment, the biocompatibility microparticle material is Cytodex-3 (AmershamBiosciences company, Piscataway, New Jersey) microcarrier, and it is comprised of the denatured collagen of with cross-linking dextran substrate, being combined.According to another embodiment, the implantable matrix of microparticles of biocompatibility comprises improvement alginate microgranule; The biocompatibility polymer is as the synthesized polymer body by hydrolytic degradation, for example many hydroxy acids such as polylactic acid, polyglycolic acid and copolymer thereof; Poe (polyorthoesters); Polyanhydride; Protein such as gelatin, collagen protein, Fibrin Glue; Or carbohydrate or polysaccharide, for example cellulose and derivatization cellulose, chitosan, alginate or its compositions.But biocompatible matrix is the material that can fade away in the time of a couple of days after the flow composition administration, several weeks or several months.The speed of degraded depends on selected biocompatible matrix, and the speed of degraded can be adjusted according to the character for the treatment of and clinical setting.

According to another embodiment, implantable matrix of microparticles can be the improvement matrix of microparticles.Can select the improvement of implantable matrix of microparticles, to optimize and/or to control the function of cell cell when implantable matrix of microparticles is combined, described cell comprises the phenotype (for example, inhibition phenotype) of cell mentioned above.According to an embodiment; improvement to implantable matrix of microparticles comprises with attachment element or adhesin polypeptide coated particle, this attachment element or adhesin polypeptide can strengthen cell suppress smooth muscle cell proliferation, reduce inflammation, increase Heparan sulfate generation, increase the generation of prostacyclin and/or increase TGF-β ₁The ability of generation.Exemplary attachment element comprises, for example fibronectin, Fibrin Glue and employing standard aqueous carbodiimide chemistry covalently bound cell adhesion part (comprising for example RGD).Other cell adhesion part comprises that having cell adhesion identifies the peptide of sequence, includes but not limited to RGDY, REDVY, GRGDF, GPDSGR, GRGDY and REDV.

According to another embodiment, implantable matrix of microparticles is the microgranule except Gelfoam.Other exemplary matrix of microparticles comprises, for example Fibrin Glue, alginate, kayexalate microcarrier, collagen coating dextran microcarrier, PLA/PGA and pHEMA/MMA copolymer (the polymer ratio of each copolymer is between 1-100%).According to preferred embodiment, these other matrix of microparticles comprise attachment element mentioned above or adhesin polypeptide after improvement.Exemplary attachment element comprises, for example gelatin, collagen protein, fibronectin, Fibrin Glue and employing standard aqueous carbodiimide chemistry covalently bound cell adhesion part (comprising RGD).Other cell adhesion part comprises that having cell adhesion identifies the peptide of sequence, includes but not limited to RGDY, REDVY, GRGDF, GPDSGR, GRGDY and REDV.

According to another embodiment, implantable matrix of microparticles through physically improved to promote cell attachment., according to an embodiment, make implantable matrix of microparticles crosslinked to strengthen its mechanical property, and promote its cell attachment and growth characteristics.According to preferred embodiment, the alginate microgranule first carries out crosslinked for the first time with calcium sulfate, then with calcium chloride, carries out crosslinkedly for the second time, and scheme is carried out routinely finally.According to another embodiment, with the source of heparin or Heparan sulfate, for example heparin-agarose adds in substrate before cell culture.

But the implantable flow composition that comprises the biocompatibility matrix of microparticles is preferably that No. 22 standard size to No. 26 standard-sized pins are sent with internal diameter.Therefore, the microgranule that forms such substrate preferably has the diameter that can pass through suitable pin hole defined herein.According to preferred embodiment, the diameter of the microgranule of such substrate is that approximately 20 μ m are to about 1000 μ m, and preferred diameter is that approximately 100 μ m are to about 500 μ m, and most preferred diameters is about 200 μ m.

Each microgranule of preferred matrix of microparticles should have at least 1 to adhere to its surperficial cell, preferably more than 1 cell.Therefore, the diameter of each microgranule should be greater than the stretching, extension diameter of selected cell type.For example, the stretching, extension diameter of endotheliocyte is about 18 μ m, and for supporting on each microgranule to adhere to the endotheliocyte more than 1, the diameter of each microgranule should be at least 20 μ m.

Preferred culture tube: in some embodiment of this paper, the about Gelfoam microgranule of 50-60mg is placed in single 50mL culture tube (Evergreen company, Los Angeles, California) with 0.2 μ m filter cap.The particle number that loses between the stage of replacement for reducing culture medium, can improve culture tube, for example, in cap district or the inside of culture tube, adds filter membrane, with the prevention microgranule, is discharged from when removing culture medium or other is unpredictably removed; Can be by culture medium but can not be by the barrier film of microgranule to adding in culture tube, with the culture medium that forms the separation that is communicated with by fluid between each several part partly and the microgranule part; Perhaps in the culture tube bottom, add and can open and emit the nozzle of culture medium under the condition that does not upset granule.

Cell is inoculated into implantable material: before the cell inoculation, add 70% ethanol in microgranule, then with PBS or HEPES, rinse for several times.Then microgranule is in antibiotic-free EGM-2, at approximately 37 ℃, 5%CO ₂Under the condition of/95% air rehydrated 12 to 24 hours.Then the about aliquot of 50-60mg microgranule is taken out from its rehydrated container, be placed in single tissue culture ware.The microgranule aliquot is with every mg microgranule 2 * 10 ³To 2 * 10 ⁴The preferred density inoculation of individual cell.The cell-matrix mixture is sucted lower several, to form the suspension of homogeneous.Then culture tube is at 37 ℃, 5%CO ₂, 90% humidity condition under regularly stir and hatch 3-4 hour, to promote cell attachment.Be about 5mg microgranule/mL culture medium with adding other 9mL EGM-2 (final culture volume be 10mL) in backward every pipe, obtaining final microgranule volume ratio.

After this, changed a subculture in every 2-3 days, until cell reaches the state of being paved with.In a preferred embodiment, cell preferably goes down to posterity 6 times, but can use passage number still less or more cell.

Cell growth curve and being paved with: respectively about the 3rd or 4 day, 6 or 7 days, 9 or 10 days and 12 or 13 days or above-mentioned time, but take out implantable flow combination matter sample and carry out cell counting, estimate cell survival rate, set up cell growth curve and analyze, with the growth characteristics of evaluation cell, and after determining whether to reach closely and being paved with, being paved with or be paved with.But the preparation that contains the implantable flow composition of Porcine aorta endothelial cells graft, its representational growth curve is seen Fig. 1.In this embodiment, the form of implantable material is microgranule.Normally, those of ordinary skills understand acceptable Growth of Cells index when early, middle and late time point, for example in early days time point (for Fig. 1, be about 3-10 days) observe the growth of cell number, then be closely to be paved with phase (for Fig. 1, be about 10-13 days), be then cell reach be paved with rear cell number stable phase (for Fig. 1, be about 13-15 days) and cell be paved with the maintenance phase (for Fig. 1, being about 15-17 days) of rear cell number.For purpose of the present invention, the preferred cell mass of at least 72 hours in stable phase.

But cell counting by with implantable flow composition aliquot with the 0.8mg/ml Collagenase solution in insulin-EDTA (being applicable to the Gelfoam microgranule), perhaps the solution of glucanase and insulin-EDTA (being applicable to the Cytodex-3 microgranule) fully decomposes and completes.But after measuring the volume of decomposed implantable flow composition, the cell suspending liquid of known volume dilutes (cell expects that to tongue blue ratio is 4: 1) with 0.4% trypan blue, and with the trypan blue exclusion method, measures survival rate.Living cells, inactivation cell and total cell are counted with hematimeter.With viable count, growth curve is set up in the natural law mapping of cultivating.After reaching and being paved with, cell betransported, and is used for transplanting.

, for purpose of the present invention, will be paved with to be defined as in every mg biocompatibility microgranule and have at least approximately 8 * 10 ³, there is total cell number approximately 7 * 10 in individual cell in preferred every aliquot 50-70mg microgranule ⁵To approximately 1 * 10 ⁶Individual, and its survival rate is preferably at least about 90% but be not less than approximately 80%.Cell survival rate preferably is at least approximately 90% but be not less than approximately 80%.If cell was not paved with after 12 or 13 days, change culture medium, continued to hatch one day.Continue this process, until reach, be paved with, perhaps until inoculate rear approximately 14 days.If determine that after the process inspection cell is paved with, carry out last culture medium and change.Last culture medium is changed and is carried out with the EGM-2 without phenol red and antibiotic-free.After culture medium is changed, immediately culture tube is set up the asepsis plug sealing cap and be used for transportation.

Evaluation of Functional: for purpose of the present invention described herein, but implantable flow composition was further tested functional labelling before transplanting.For example, collection condition culture medium in the training period, to determine the Heparan sulfate, the transforming growth factor β that are produced by the endotheliocyte of cultivating ₁(TGF-β ₁), basic fibroblast growth factor (b-FGF) and nitric oxide production level.In some preferred embodiment, when total cell number is at least about 2 * 10 ³Individual cell/cm ³, preferably at least about 8 * 10 ³Individual cell/cm ³, and living cells percent is at least about 80-90%, is preferably greater than and equals 90%, most preferably at least about 90% the time, but implantable flow composition can be used for purpose described herein.Heparan sulfate in conditioned medium is at least about 0.5-1.0 μ g/10 ⁶Individual cell/sky, preferably at least about 1.0 μ g/10 ⁶Individual cell/sky.TGF-β in conditioned medium ₁Be at least about 200-300picog/ml/ days, preferably at least about 300picog/ml/ days; B-FGF in conditioned medium, lower than about 200picog/ml, preferably is no more than approximately 400picog/ml.

The level of Heparan sulfate can adopt conventional Dimethylmethylene blue-chondroitinase ABC digestion photometry to carry out quantitatively.Total sulphation glycosaminoglycans (GAG) level is measured with Dimethylmethylene blue (DMB) dye binding method, the aching and limp ossein of bright sulfur that the method is collected culture medium (collection media) dilution known quantity by use obtains standard curve, then unknown sample and standard curve is compared.Before adding the DMB developer, other sample of conditioned medium is mixed with chondroitinase ABC, to decompose chrondroitin and dermatan sulfate.All light absorption values are all at the maximum absorption wavelength of the DMB dyestuff that is mixed with the GAG standard substance---and be generally the 515-525nm left and right and measure., by deduct the concentration of chrondroitin and dermatan sulfate with the total sulphation glycosaminoglycans concentration in the conditioned medium sample, calculate every 10 ⁶The Heparan sulfate level of individual cell every day.The activity of chondroitinase ABC is proved conclusively by decomposing the aching and limp ossein sample of bright sulfur.If the aching and limp ossein of bright sulfur that decomposes is lower than 100%, suitable correcting condition media samples.The level of Heparan sulfate can also adopt the ELISA method to carry out quantitatively with monoclonal antibody.

TGF-β ₁Adopt the ELISA method with the level of b-FGF, with monoclonal antibody or polyclonal antibody, preferred polyclonal antibody carries out quantitatively.Collect culture medium (collection media) contrast and carry out quantitatively with the ELISA method equally, and the TGF-β of suitable correcting sample in the culture medium contrast ₁With the b-FGF level.

Nitric oxide (NO) level is quantitative with standard Griess reaction method.Nitric oxide production transition and volatility make it be not suitable for most of detection methods.Yet, nitric oxide production two stable catabolites---nitrogen peroxide (NO ₃) and nitrogen dioxide (NO ₂) can detect with conventional photometry.The Griess reaction method is nitrogen dioxide with nitrogen peroxide through enzymatic conversion method under nitrate reductase exists.Nitrogen dioxide is to absorb the approximately form that the azo-dye product is arranged of the visible light at 540nm scope place, through colorimetric determination.The nitric oxide production level that exists in system is by being converted into nitrogen dioxide with all nitrogen peroxides, measure the nitrogen dioxide total concentration in unknown sample, the content of nitrogen dioxide that then will obtain is converted into nitrogen peroxide with known quantity the standard curve that produces after nitrogen dioxide and compares.

Aforementioned preferred inhibition phenotype is with above-mentioned Heparan sulfate, TGF-β ₁, NO and/or b-FGF quantitative analysis, and the external quantitative analysis of following smooth muscle cell growth and thrombosis suppression ratio is evaluated.For purpose of the present invention, when one or more these other analyzed in vitro proves, but when implantable flow composition demonstrates preferred inhibition phenotype, but this implantable flow composition can be prepared for transplanting.

, for the inhibition of in-vitro evaluation to smooth muscle cell growth, measured the inhibiting value relevant to the endotheliocyte of cultivating.With in pig or the sparse smooth muscle cell growth culture medium that is inoculated in 24 hole tissue culturing plates of human aortic smooth muscle cell (SmGM-2, Cambrex BioScience company).Made cell attachment 24 hours.Then this culture medium is replaced with the smooth muscle cell basal medium (SmBM) that contains 0.2%FBS, cultivate 48-72 hour, Growth of Cells is stagnated.To be paved with rear Endothelial cell culture thing and dilute twice with the SMC growth medium by 1: 1, and make conditioned medium, and it is added in the Smooth Muscle Cell thing.Each test includes the positive control that suppresses smooth muscle cell growth, for example heparin.After 3-4 days, with the Coulter enumerator, the cell number in every duplicate samples is counted.Add conditioned medium before and be exposed to conditioned medium and control medium (standard growth culture medium by relatively facing, add or do not add somatomedin) lower every hole smooth muscle cell number after 3-4 days, determine the effect of conditioned medium to smooth muscle cell proliferation.The inhibiting value that inhibiting value that will be relevant to the conditioned medium sample and positive control are relevant compares.According to preferred embodiment, if the suppression ratio of conditioned medium reach heparin contrast suppression ratio approximately 20%, but think that this implantable flow composition has inhibition.

, for the suppression ratio of in-vitro evaluation to thrombosis, measured the level of the Heparan sulfate relevant to the endotheliocyte of cultivating.Heparan sulfate has antiproliferative and antithrombotic property.The conventional Dimethylmethylene blue that the employing above-detailed is crossed-chondroitinase ABC photometry or ELISA method, calculate every 10 ⁶The Heparan sulfate concentration of individual cell.Heparan sulfate in conditioned medium is at least about 0.5-1.0mg/10 ⁶Individual cell/sky, preferably at least about 1.0mg/10 ⁶During individual cell/sky, but this implantable flow composition can be used for purpose described herein.

Another method of estimating the thrombosis suppression ratio is included in the inhibiting value of the external test pair platelet aggregation relevant to platelet rich plasma.At room temperature add sodium citrate in the pig blood sample, obtain porcine blood plasma.Sodium citrate blood plasma is centrifugal under gentle rotating speed,, so that erythrocyte and leukocyte are agglomerating, stays platelet suspension in blood plasma.With being paved with rear Endothelial cell culture thing preparation condition culture medium, and add in the platelet rich plasma aliquot.Add platelet aggregating agent (agonist) in contrast in blood plasma.Platelet agonist generally comprises arachidonic acid (arachidonate), ADP, collagen protein, epinephrine, RCT (ristocetin, can available from Sigma-Aldrich Co. company, Saint Louis, the Missouri State).Another part do not add the blood plasma aliquot of platelet agonist or conditioned medium to be used for the spontaneous platelet aggregation of establishment of base line.Each test also comprises the positive control of anticoagulant.Exemplary positive control comprises aspirin, heparin, abciximab (ReoPro

, Eli Lilly company, Indianapolis, the state of Indiana), tirofiban (Aggrastat

, Merck ﹠amp; Co., Inc. company, Whitehouse Station, New Jersey) or Eptifibatide (Integrilin , MillenniumPharmaceuticals, Inc. company, Cambridge, Massachusetts).Then measure with the gathering instrument platelet aggregation that obtains under all test conditions.Assemble instrument and measure platelet aggregation by monitor optical densities.Work as platelet aggregation, have more light can pass through sample.Assemble function " platelet aggregation unit " expression of the result of instrument report with platelet aggregation speed.Measure maximum the gathering while adding after agonist 6 minutes.By the baseline platelet aggregation of platelet rich plasma before adding conditioned medium relatively be exposed to after conditioned medium the platelet aggregation that reaches positive control, determine the effect of conditioned medium to platelet aggregation.Result represents with the percent of baseline.The inhibiting value that inhibiting value that will be relevant to the conditioned medium sample and positive control are relevant compares., according to preferred embodiment, when approximately 20% time of the suppression ratio of the positive contrast of suppression ratio of conditioned medium, but can think that this implantable flow composition has inhibition.

Cask: after culture medium is changed, but implantable flow composition is packed to be used for transportation immediately.According to an embodiment, the same culture tube that uses during cultured cell on implantable material is set up the asepsis plug sealing cap and is used for transportation.For being reduced in the risk of microgranule between last flush period and/or cell decant, can improve so that it comprises cask, for example, having can be by culture medium and rinse solution, but can not decant goes out filter or other acquisition equipment in the aperture of granule and/or cell.

If but implantable flow composition transports in containing blood serum medium, but rinse implantable flow composition before facing transplanting, preferably rinse in culture tube.Then remove filter or other acquisition equipment from cask, with being used in cell transplantation microgranule inhalation syringe, send.Unnecessary rinse solution in the exhaustjet device, be connected to syringe with pin, conduit or other delivery apparatus, is used for sending to therapentic part.Then according to for example a kind of in exemplary medication hereinafter described, but flow composition is delivered to the patient., if but implantable flow composition transports in serum-free medium, in clinical place, need not to carry out last cleaning procedure.

According to another alternate embodiment, but implantable flow composition is drawn to syringe from culture tube, adds the 1-20ml culture medium, or the culture medium of enough volumes is transported together and preserves, add the cap sealing to syringe, this material of transportation in the syringe of sealing.At implant site, unnecessary culture medium is discharged from syringe.Then pin, conduit or other delivery apparatus are connected to syringe, are used for sending to therapentic part.According to this embodiment, but flow composition directly is delivered to the patient from the transportation syringe.

Implantable compositions of the present invention can be supplied in the end product container, comprise 50ml or 60ml seal tissue culture vessel or the preload syringe for example through filter cap, improved, described end product container all preferably contains the 50-60mg microparticle material of having an appointment, at the about 45-60ml of every equal portions, the endotheliocyte of implanting in the endothelial cell growth culture medium of preferred approximately 50ml adds up to approximately 7 * 10 ⁵To approximately 1 * 10 ⁶Individual.Total cell number that each patient accepts is preferably approximately 0.6 * 10 ⁴-12 * 10 ⁴Individual cell/kg body weight, but be not less than 2 * 10 ³Individual cell/kg body weight also is not more than 2 * 10 ⁵Individual cell/kg body weight.

As this paper expection, material of the present invention comprises cell, preferred vascular endothelial cell; In a preferred embodiment, the preferred survival rate of described cell is approximately 90%, and preferred density is approximately 1.4 * 10 ⁴-2.1 * 10 ⁴Individual cell/mg microgranule; When being paved with or closely be paved with, the heparin that the conditioned medium that described cell produces comprises is at least about 0.5-1.0mg/10 ⁶Individual cell/sky, preferably at least about 1.0mg/10 ⁶Individual cell/sky.TGF-β in conditioned medium ₁Be at least about 200-300picog/ml/ days, preferably at least about 300picog/ml/ days; B-FGF in conditioned medium, lower than about 200picog/ml, preferably is not more than approximately 400picog/ml.

According to another embodiment, but added one or more other materials in flow composition before administration.These materials include but not limited to that antiinflammatory, glycosaminoglycans, prostaglandin, prostaglandins, cytokine include but not limited to TGF and VEGF, angiotensin and related compound, tyrosine kinase inhibitor, immunosuppressant, vitamin, glucocorticoid, antioxidant, free radical scavenger, peptide hormone, angiogenesis factor and Angiostatin.

Carrier: according to an embodiment, but implantable flow composition can be chosen wantonly and comprises carrier fluid.Preferred carrier fluid is the cell growth medium that makes administration convenient.But the administration of the implantable flow composition of laboratory simulation shows, but carrier is not to keep the essential condition of cell integrity while processing flow composition.Be unexpectedly, can not have as described herein carrier or other to be commonly used to the reagent of Cell protection integrity in cell preparation, or be commonly used to improve the reagent of processing and reducing the non-solid preparation division of maybe can flowing that is caused by shearing force.

Yet, carrier can be for example, during cell culture, comprise the discharge conditioned medium, while importing new culture medium, but at the implantable flow composition of transportation pre-treatment, when this material inhalation delivery is used syringe, and/or this material is discharged and with this material delivery during to perivascular canal or other target site from syringe, but be used for improving mobility and the survival rate of cell at flow composition.

During carrier can import implantable material in the different time points of cell culture program.For example, in the cell culture program, carrier can be when microgranule hydration (cell inoculation before), be about to carry out the cell inoculation before or add matrix of microparticles when carrying out the cell inoculation, and/or add with incremental mode when predetermined culture medium replacing.By at cell culture, introducing in early days carrier, those cells that may be adversely affected can be discarded, remaining cell can breed to keep enough cell masses.

In addition, carrier can reach and be paved with or closely be paved with at cell, while carrying out last culture medium replacing, and is about to transplant front introducing.Yet, observe and added undiluted glycerol carrier may cause cell shock before facing administration, thereby cell is suffocated fully and reduce the cell survival rate of expection and lower than the usefulness of acceptable level.Based on these observed results, but to disposable high viscosity carrier fluid such as the glycerol of all adding in flow composition, likely cell is produced harmful effect, and should avoid.

, for estimating the effect of other candidate's carrier fluid, adopt high microgranule to carry out preliminary test to the carrier ratio, the minimum of required carrier while with mensuration, producing a desired effect.The Expected Results of estimating comprises, for example improves and processes and improve the mobility of compositions and keep survival rate and the effect of cell.In addition, but also study to test the ability that the implantable compositions of plane is converted into implantable particle type flow composition under the condition that does not affect cell survival rate or function.For example, in initial test solution, the 50mg microgranule only contains the carrier solution of the 0.1-1% that has an appointment.This concentration increases to maximum take incremental mode and contain approximately 10% carrier in the 50mg microgranule.

In the test of carrying out subsequently, carrier fluid during from the inoculation of microgranule hydration or cell, add incrementally in whole cultivation process, be paved with and prepare transportation and/or while bestowing the patient with regard to making when but the cell in implantable flow composition reaches like this, the concentration of the carrier fluid in implantable material during from early stage cell attachment the acceptable carrier fluid the low ratio of microgranule has been increased to higher desired proportions.For example,, according to exemplary scheme, when initial hydration and every subculture replacing subsequently, 0.1% carrier solution is joined in microparticle material, until the acquisition ultimate density is 1% carrier solution.We think, although all can have some cells to run off each while introducing carrier fluid, but most cells sneak in the implantable flow composition of vehicle treated, and can grow to and be paved with or closely be paved with under this environment.

Carrier includes but not limited to that rare glycerol, alginate (preferred approximately 1% alginate soln), glucosan or dextrose (glucosan of preferred approximately 1-10% or dextrose solution), other sugar comprise for example glucose, sucrose and fructose, starch (preferred approximately 6% hydroxyethyl starch solution), gelatin (gelatin solution of preferred approximately 1-2%), endothelial cell growth culture medium, endothelial basal medium, other cell growth medium or neutral buffered salt.In the unspecified solution percentage range of other this paper is also included within.The change of carrier partly depend on biocompatible matrix used type, be transplanted on material cell type and for sending selected mode of administration.For example, be used for the cell culture medium that initial hydration and culture medium are subsequently changed, according to the carrier of selecting, can comprise by volume approximately 0.5% to about 10% carrier fluid.Normally, selected carrier should be under dosage used and concentration no cytotoxicity, and has sufficient permeability, to allow air and nutrient substance to flow into (with the sustenticular cell growth) and to flow out (to discharge the cell waste product) implantable compositions.

The impact of undiluted glycerol carrier on cell survival rate: Porcine aorta endothelial cells is inoculated on 100mg Gelfoam microgranule, and propagation is to being paved with.But collect the particle type flow composition, and be placed in the 50ml test tube and mix with the undiluted glycerol aliquot of 3ml, then be placed in the syringe that is connected with 24,26 or No. 27 pins.Content in all syringes is slowly discharged by the pin that connects, and is collected in the 50ml test tube.Only there is the cell of 0-5% to survive afterwards in discharge in all living cells (cell death of 95-100%), the cell survival rate that has 86-93% with the identical test that does not add glycerol is compared, and has shown that obviously this carrier fluid adds fashionable adverse effect to cell survival rate as single dose to being paved with cell in undiluted situation.Not yet determine at present to use this carrier fluid whether to be conducive to keep being paved with of cell monolayer.

The extruding of plane material and modification:, according to another embodiment, after cell is implanted into implantable compositions fully and reaches and be paved with, solid, semisolid or major diameter compositions are modified, to form the microgranule that can send by the injection delivery apparatus.According to an embodiment, cell is cultivated as described in detail above like that under carrier fluid exists, with being paved with and integrity of cell during keeping material and modifying.

An embodiment according to method of modifying,, in case cell reaches and is paved with on the implantable compositions of plane, be transferred to syringe with compositions.For adapting to this plane form, syringe can be with pin or with large vent needle.Syringe drinks up the elastic linear form, then, under pressure, implants the compositions of cell and discharges by syringe port, but form nonplanar flow composition., in order to obtain preferable particle size, may need repeatedly to pass through from syringe.For example, material can, first by there is no the syringe of pin,, then by large vent needle, then pass through the more pin of aperture, until material reaches particle diameter and the mobility of expection.Multiple through then out and the increment of particle diameter is modified the injury amount that can advantageously reduce during such material is modified cell, and keep the degree of being paved with of cell.

The operation sealant: in some other embodiment, but flow composition of the present invention usually can also be in addition especially as anastomotic stoma sealant, perhaps surgical sealants.In so dual-purpose embodiment,, when with the outer surface of tubular structure, contacting, perhaps with arc, be applied to outer surface, while perhaps along the circumferential direction using, the abutment of all right two or more tubular structures of effective sealing of compositions, the perhaps hole in the sealed tubular structure.Such sealant can be eliminated the needs of sewing up, and described stitching can for example further damage vascular tissue and impel the chamber endothelial injury.Such sealant can also provide extra stability near anastomotic stoma, thereby strengthens the effect of any suture repair.All essential conditions are that the functional expression simultaneously based on cell of expection phenotype and the said composition of cell is not disturbed or weakened to the closed type function of this dual purpose compositions or characteristic.

Purpose for some sealant embodiment, but flow composition comprises the biocompatibility substrate, this substrate self comprises having the sealant characteristic, also has simultaneously the composition that supports endothelium or the needed characteristic of endothelioid cells group, such as but not limited to the fibrin net.Similarly, biocompatibility substrate itself can have simultaneously the sealant characteristic and support the needed characteristic of cell mass.In the situation that other embodiment, the functional of sealant can be provided by cell at least in part.For example, the cell that expection is combined with compositions produces can modify the material of substrate, thereby makes substrate obtain the sealant characteristic, also shows simultaneously/keep the cell function that it is essential.Some cell can produce such material naturally, and other cell can be by producing such material after transformation.

But the storage life of implantable flow composition: comprise that but the implantable flow composition that is paved with, closely is paved with or is paved with rear cell mass can at room temperature keep stable and at least two weeks of existing state.But the implantable flow composition of this class preferably, at about 45-60ml, is more preferably from about preserved in the transportation culture medium that contains or do not contain extra FBS of 50ml.The transportation culture medium comprises and does not contain phenol red EGM-2 culture medium.Transportation can add FBS in culture medium, until reach FBS, is about by volume 10%, and perhaps the FBS total concentration is approximately 12%.Yet, due to before transplanting, but FBS must remove from implantable flow composition, the amount of the FBS that therefore uses in preferred restriction transportation culture medium, to reduce before transplanting the washing time that needs.

But the freezing preservation of implantable flow compositionBut: comprise the implantable flow composition that is paved with, closely is paved with or is paved with rear cell mass can freezing preservation with storage and/or be transported to Clinical Institutions, and do not reduce its clinical efficacy or integrity after last thawing.But implantable flow composition is preferably at 15ml cryovial (Nalgene

, Nalge Nunc Int ' l company, Rochester, New York) in, containing the 5ml CryoStor CS-10 solution (BioLife Solutions company, Oswego, New York) of having an appointment, approximately 10%DMSO, approximately 2-8% glucosan and approximately freezing preservation in the solution of 50-75%FBS.Cryovial is placed in the cold isopropanol water-bath, is transferred in-80 ℃ of refrigerators 4 hours, then be transferred in liquid nitrogen (150 to-165 ℃).

The approximately 15min that then but the aliquot of the implantable flow composition of freezing preservation at room temperature slowly thawed, then thawed approximately 15 minutes again in room-temperature water bath.Then with material with about 15ml washing with culture medium (wash media) washing approximately 3 times.Washing with culture medium comprise contain or do not contain 50 μ g/ml gentamycins without phenol red EBM.Initial twice cleaning procedure at room temperature carried out approximately 5 minutes.Last cleaning procedure is at 37 ℃, 5%CO ₂Under carried out approximately 30 minutes.

Thaw and cleaning procedure after, the material that makes freezing preservation dormancy approximately 48 hours in the recovery liquid of about 10ml.For the pig endotheliocyte, recovering liquid is at 5%CO under 37 ℃ ₂In the EBM-2 that is added with 5%FBS and 50 μ g/ml gentamycins.For human endothelial cell, recovering liquid is antibiotic-free EGM-2.The rear adjusting of further thawing can used and/or pack for storage or before transporting and carried out other at least 24 hours.

Loading in delivery apparatus and picked-up: such as described in detail above, but the aliquot of flow composition is in culture tube or syringe, at about 45-60ml, the transportation culture medium intermediate package and the transportation that contain or do not contain serum of preferred approximately 50ml, sustenticular cell reaches 14 days under the condition of not changing culture medium.Before transplanting, decant goes out unnecessary culture medium, and, if having serum in the transportation culture medium, but with implantable flow composition rinsing for several times, to remove all residual serum.Because but the preparation of the particulate form of some implantable flow composition is easy to disperse, and therefore lost cell is paved with state, so in last cleaning procedure, said composition should be retained in cask.In addition, but to the improvement of cask, preferably can keep in operation the improvement of the integrity of implantable flow composition, described improvement includes but not limited to filter and/or independent cultivation compartment.

But rinse implantable flow composition for several times after, but the flushing liquor that keeps about 1-3ml is at the top of implantable flow composition, to be conducive to absorbing to delivery apparatus this material for example in syringe.Then operate delivery apparatus, but with in implantable flow composition inhalation delivery device, as far as possible careful during operation, the destruction that is paved with cellular layer is minimized.According to another embodiment, use the loading attachment of material, for example at the cask opening with send with the infundibulate interface between syringe, material cell breakage in being transferred to the process of delivery apparatus is reduced.To delivery apparatus, the about 1ml of discharge section remaining liq,, to be ready to delivery apparatus, fill up voidage from delivery apparatus in material transfer.But the approximately surplus flushing liquor that 0-2ml is arranged in being delivered to patient's implantable flow composition.Prepare now but implantable flow composition is delivered to therapentic part.

Pass through pin: for proving practicality of the present invention, but an implantable flow composition of preferred particle type (implanting the HAE of Gelfoam microgranule) has passed through No. 22 pin holes (internal diameter=0.016 inch).In addition, the scope of the pin of other preferred composition PAE of microgranule (implant Gelfoam) pin hole that can pass through be approximately No. 21 pins (internal diameter=0.019 inch) to No. 28 pins (internal diameter=0.007 inch).

As shown in table 1 below, embodiment of the present invention can be under cell number, survival rate or the functional condition that has no adverse effects of the cell to by pin, by the pin of internal diameter in these scopes.Surprisingly, cell preparation can do not affect or damage under the state of being paved with or functional condition picked-up and from scope be No. 21 (internal diameter=0.019 inch) to No. 30 (internal diameter=0.006 inch), flow out in the pin hole of preferred No. 24 (internal diameter=0.012 inch).Equally surprisingly, cell is after passing through pin, and the cell growth medium of only using is very good as performance under the condition of carrier fluid.

As mentioned below, cell mixes with matrix of microparticles, and propagation is to being paved with.Be paved with rear 3 days, but with the flow composition that obtains by inside diameter ranges be approximately 0.007 inch to the about sterile needle of 0.018 inch, collect, recovery is 48 hours in endothelial cell growth culture medium-2 (EGM-2).After convalescent period, made culture medium conditionization 24 hours.Then the conditioned medium by rear cell is estimated.Conditioned medium after passing through is estimated its product basic fibroblast growth factor (b-FGF), Heparan sulfate (HS) and transforming growth factor β ₁(TGF-β ₁) acceptable level.In addition, carry out smooth muscle cell and test to show the inhibitory action of culture medium to smooth muscle cell proliferation.The experimental result of the compositions by pin and the sample by pin are not compared.

Table 1

By No. 22 pins

Experimental result	No. 22 pins	Needleless
			Cell counting+survival rate	6.4×10 6+88.3％	7.95×10 6+90.8％
μg HS/10 6Individual cell	1.7	1.0
			pg TGF-β 1/10 6Individual cell	488.6	575.3
pg bFGF/10 6Individual cell	227.6	229.2
			Smooth muscle cell inhibitory action (PCCM/ effects of heparin effect ratio)	75％	100％

By No. 24 pins

Experimental result	No. 24 pins	Needleless
			Cell counting+survival rate	1×10 6+86％	2.78×10 6+92.6％
μg HS/10 6Individual cell	9.33	8.6
			pg TGF-β 1/10 6Individual cell	545.7	571.4
pg bFGF/10 6Individual cell	2491	744
			Smooth muscle cell inhibitory action (PCCM/ effects of heparin effect ratio)	65％	45％

Pass through conduit: but in another demonstration of the unexpected characteristic of flow composition of the present invention, preferred formulation is by syringe and/or penetrate instrument, and for example wire guide loads and administration (table 2).In a research, wire guide is and thin-walled Nitinol pin (Nitinol needle, external diameter=No. 24 standard size is arranged; Internal diameter=No. 22 standard size) 6French conduit (TransVascular Corp. company, Palo Alto, California).

But with flow composition by the wire guide of internal diameter in this scope to by after cell number, survival rate or the bio of cell go out not have harmful effect.According to preferred embodiment, cell is inoculated on matrix of microparticles, and propagation is to being paved with.Be paved with rear 3 days, but with flow composition by inside diameter ranges be approximately 0.007 inch to the about sterile needle conduit of 0.018 inch, collect, recovery is 24 hours in endothelial cell growth culture medium-2 (EGM-2).After convalescent period, made culture medium conditionization 24 hours.Then the conditioned medium of the cell by after pin is estimated.Conditioned medium after passing through is estimated its product basic fibroblast growth factor (b-FGF), Heparan sulfate (HS) and transforming growth factor β ₁(TGF-β ₁) acceptable level.In addition, carry out smooth muscle cell and test to show the inhibitory action of culture medium to smooth muscle cell proliferation.The experimental result of the cell by pin and the result of the cell of the implantation microparticle material sample by wire guide are not compared (in Table 2).

Table 2

Pass through wire guide

Experimental result	Wire guide	Without wire guide
			Cell counting+survival rate	7.9×10 6+91.2％	9.9×10 6+91％
μg HS/10 6Individual cell	1.1	1.0
			TGF-β 1 pg/m	360	336
bFGF pg/mL	58	61.7
			Smooth muscle cell inhibitory action (PCCM/ effects of heparin effect ratio)	92％	85％

Intravascular administration: but flow composition can be at intracavitary administration, that is, and intravascular administration.For example, compositions can be sent by any device that is treated blood vessel that can insert.Endoscope navigation system can be used for delivery apparatus is positioned medicine-feeding part, comprises for example fluoroscopy and/or other endoscope navigation system well known in the art of intravascular ultrasound (IVUS), color doppler ultrasonography, dual-functional ultrasonic, other conventional Ultrasound, angiography, nuclear magnetic resonance (NMR) vessel visualization (MRA), NMR (Nuclear Magnetic Resonance)-imaging art (MRI), CT scan, evaluation backing positions.In addition, medicine-feeding part can be located by palpation.Preparation can carry out separately to the endovascular delivery of medicine-feeding part, and perhaps with other blood vessel internal program, for example the transplanting of balloon angioplasty or support or other device combines, before other blood vessel internal program, simultaneously or carry out afterwards.

In one embodiment, be equipped with and cross or penetrating device on the intraluminal delivery device, this device can pass the lumen of vessels wall, arrives the surface, non-chamber of blood vessel.Then but flow composition is at impaired or ill target site or in the adjacent position at this position or near the non-chamber surface deposition of the blood vessel this position.

The surface, non-chamber (also being called outside chamber) that this paper relates to can comprise outer surface or the circumvascular surface of blood vessel, perhaps can be in the adventitia of for example blood vessel, middle film or inner membrance.For purpose of the present invention, outside non-chamber or chamber, position is any surface except the inner surface in chamber.

The penetrating device that this paper relates to allows for example single delivery sites, a plurality of delivery sites of perhaps with the expection geometric configuration, arranging, but complete sending of flow composition surface, vasotropic non-chamber under the condition of not disturbing impaired or ill target site.A plurality of delivery sites can be by arrangements such as circle, buphthalmos shape (bulls-eye) or line array.Penetrating device can also be the form of support perforator, such as but not limited to the sacculus support that comprises a plurality of delivery sites.

Percutaneous dosing: for purpose of the present invention, but flow composition usually at the position of needs treatments, the position adjacent with this position or near the position topical this position.But the position of flow composition deposition is outside chamber.As this paper estimated, the local deposits outside chamber can as mentioned belowly be completed via skin.

But flow composition can be used pin, conduit or other suitable delivery apparatus dermal delivery.But in the dermal delivery flow composition, can use orientation method, make to the sending of position of needs treatment and be more prone to.Orientation step is chosen wantonly.Endoscope navigation system can be used for the position of chamber external administration is positioned, and comprises for example fluoroscopy and/or other endoscope navigation system well known in the art of intravascular ultrasound (IVUS), color doppler ultrasonography, dual-functional ultrasonic, other conventional Ultrasound, angiography, nuclear magnetic resonance (NMR) vessel visualization (MRA), NMR (Nuclear Magnetic Resonance)-imaging art (MRI), CT scan, evaluation backing positions.In addition, medicine-feeding part can be located by palpation.After intravasation week gap, but the doctor is deposited on position chamber outside with flow composition, and this chamber outside, position is on the position that needs are treated, or the position for the treatment of with needs is adjacent, or near the position that needs are treated.Orientation or authentication step can at random be carried out, and are not that enforcement method of the present invention is necessary.

In another embodiment, but implantable flow composition position local delivery outside the chamber of surgical exposure, and outside this chamber, position is on the position of needs treatments, or adjacent with the position of needs treatment, or near the position of needs treatment.Complete at the position that the orientation of sending in this case, and guide need to be treated by direct observation.Equally in this case, can use simultaneously authentication step mentioned above to help to send.In addition, authentication step is chosen wantonly.

Medicine-feeding part: according to the preferred embodiments of the invention, penetrating device inserts with endovascular delivery device intravascular surface of internal cavity, perhaps inserts by surrounding tissue in dermal delivery.Administration can be pointed to the proximal location of impaired or ill target site, impaired or the remote location of ill target site or the position of impaired or ill target site.In some clinical experimenters, insert penetrating device at impaired or ill target site and may disturb impaired or ill target site.Therefore, in this class experimenter, care should be used to ground is inserted in penetrating device with impaired or ill target site and has on the position of certain distance, and preferred distance is determined according to particular case at the moment by the clinician.

But the flow composition preferred deposition is on circumvascular surface, and this surface is positioned on the impaired or disease sites that is treated, or impaired or disease sites is adjacent with this, or at this near impaired or disease sites.Compositions can be in the various positions deposition relevant to impaired or ill target site, for example, on impaired or ill target site, on the position adjacent with impaired or ill target site, for example in the upstream of impaired or ill target site, at the reverse blood vessel outer surface of impaired or ill target site.According to preferred embodiment, adjacent regions be from impaired or ill target site approximately 2mm to the about position of 20mm.In other preferred embodiment, deposition site at about 21mm to about 40mm.In a further preferred embodiment, deposition site at about 41mm to about 60mm.In a further preferred embodiment, deposition site at about 61mm to about 100mm.In addition, adjacent regions is any other adjacent position of being determined by the clinician, and the compositions that deposits in this position can show expected effect.

According to an embodiment, but before implantable flow composition administration, make plane administered area arbitrarily in target site outside chamber.But the plane administered area is the zone that the implantable flow composition of certain volume is accepted in preparation, can adopt for example blunt dissection, sacculus post-mortem method, fluid post-mortem method or other anatomy manufacturing well known in the art.Administered area can the interior or blood vessel dissection device manufacturing on every side with blood vessel.But making administered area makes flow composition easier in the implantation of target site.Administered area is not that enforcement is essential to the invention.

But implantable flow composition can be with multiple configuration in the medicine-feeding part administration.For example, but the administration of implantable flow composition can be with linear applications, and is parallel with blood flow direction; Along the circumferential direction application, vertical with blood flow direction; Or at medicine-feeding part, be block.But sending of aforementioned dissection step and flow composition also can occur simultaneously or occur sequentially.For example,, if send under pressure, but just can being used for fluid, implantable flow composition self dissects.Yet, but there is the risk by the tissue injury of pressurized delivery flow combination deposits yields in this anatomic method, but and the pressurization of implantable flow composition may destroy the degree of being paved with of cell during by perivascular canal.In addition, delivery apparatus can be inserted the chamber external series gap to complete blunt dissection, when delivery apparatus is recalled from the plane administered area of new generation, but namely complete the administration of implantable flow composition.

Angiography: according to the preferred embodiments of the invention, site-specific delivery of drugs position under the assistance of navigation system.According to an embodiment, navigation system is endoscope navigation system, for example intravascular ultrasound (IVUS).Intravascular ultrasound provides 360 ° of transverse section images of lumen of vessels, comprises surrounding structure and blood vessel.

In a further preferred embodiment, endoscope navigation system is angiography.In certain embodiments, employing for example contrasts angiography contrast medium is added in the microgranule cell suspending liquid, makes the penetrating device imaging, determines the position of penetrating device, and the microgranule cell suspending liquid is placed in patient body.

The endoscope navigation system at other positioning chamber external administration position includes but not limited to fluoroscopy and/or other endoscope navigation system well known in the art of color doppler ultrasonography, dual-functional ultrasonic, other conventional Ultrasound, nuclear magnetic resonance (NMR) vessel visualization (MRA), NMR (Nuclear Magnetic Resonance)-imaging art (MRI), CT scan, evaluation backing positions.In addition, medicine-feeding part can be located by palpation.

Adopt for example angiography or IVUS, but make implantable flow composition in video picture in the external series gap of chamber after administration.According to an embodiment, but after carrying out administration, video picture has been delivered to chamber external series gap rather than intracavity gap to guarantee implantable flow composition.

Dosage: in a preferred embodiment of the invention, but each flow composition that can send administration contains and has an appointment 1 * 10 in the volume of the about implantable microgranule of 50-60mg ⁶Individual cell.But the single-dose of flow composition or multiple dosing can be sent to single therapentic part.But the multiple dosing of flow composition can be sent to single patient.The excursion of multiple dosing comprises to single therapentic part carries out multiple dosing, to a plurality of therapentic parts, carries out the single or multiple administration, and/or even provides administration in long therapeutic procedure during single treatment event.

But the administration of each flow composition includes voidage, and voidage is to stay the part of the appointment administration volume in delivery apparatus after administration.But the scope of voidage be loaded into delivery apparatus the flow composition volume approximately 1% to approximately 50%.Consider voidage, the amount of the cell of delivery apparatus and microgranule that is loaded into is greater than the actual dosage that is intended to bestow the patient.Certainly, but voidage and the thing followed depend on selected delivery apparatus to the adjustment of the volume of the flow composition that is loaded into delivery apparatus.

According to preferred embodiment, but each flow composition that can send administration is all with the packaged of single-dose.When needs were implemented multiple dose administration to single therapentic part, the dosage of expection can be loaded into single delivery apparatus, and bestows in single-dose.Yet, in the time need to implementing multiple dose administration to a plurality of therapentic parts of single patient, preferably use the aliquot of the fixing product sent of many umbers amount, repeatedly independently complete heavy dose of administration in administration.The advantage of fixedly administration aliquot comprises, reduces that to be divided into a plurality of low dose of dosage of introducing by a heavy dose inaccurate; The dosage that minimizing is introduced by the limitation of the intrinsic measurement of delivery apparatus is inaccurate; And the dosage that reduces the excessive introducing of volume of the transportation culture medium that is needed by a large amount of compositions aliquots changes.In addition, a large amount of medicaments are divided into less aliquot require compositions is carried out unnecessary processing, comprise and destroy adjacent granule, destroy and be paved with cell monolayer and other damages that cause cell.

But the multiple dosing of implantable flow composition can provide in long-term therapeutic process.According to preferred embodiment, but the predose of implantable flow composition bestow when treatment or blood vessel are got involved for the first time, carried out one time minimally-invasive administration in after this every 1-3 month, the time administration of the needs of perhaps determining the clinician.

Reflux: carry out preclinical study with the tested body of single pig of accepting Gelfoam microgranule intravascular administration.The suspension of hydration Gelfoam microgranule, with after contrast medium mixes, is loaded in the delivery machine based on conduit, conduit is inserted the blood vessel structure of tested body.Guide conduit into therapentic part, pin inserts perivascular canal by blood vessel wall, and suspension is injected perivascular canal.Injecting program and down-stream are with contrasting the angiography video picture.

The contrast angiographic results shows, when injection or administration and remove pin after, suspension returning does not all occur to Endovascular.In addition, the Histological evaluation that is treated tissue slice to through the dyeing of VerhoeffShi elastin laminin staining that carries out does not subsequently show that suspension overflows the intravasation chamber or enters the evidence of surrounding tissue from adventitia.

But the implantable flow composition of therapeutic dose, approximately 0.1ml is to about 2ml, but can be enough at the pressure of perivascular canal cause implantable flow composition to inject in the perivascular canal of single injection site before passing back into lumen of vessels.

Embodiment 1: the animal blood vessels Intervention studies

The present embodiment provides test and the testing program of using the preferred embodiments of the invention with the generation of the minimizing clinical sequela relevant with the blood vessel intervention in the tested body of animal.Adopt SOP, by at femoral artery place percutaneous balloon angioplasty and Stent Implantation, causing the exocoel surface damage.Then but implantable flow composition of the present invention is deposited in the perivascular canal adjacent with the position of angioplasty and stent in the treatment blood vessel; The details of an exemplary program is set forth hereinafter.As previously mentioned, but implantable flow composition to insert with preparation can be miscellaneous.

This research specifically comprises 26 pig experimenters that accept percutaneous balloon angioplasty and Stent.Carry out traditional percutaneous balloon angioplasty and Stent program according to the standard operation technology.Complete angioplasty and stenting, and setting up by after the blood flow that is treated blood vessel, but with implantable flow composition the position that balloon dilatation and stenting inserts and on every side of being applied to as mentioned below.

Operative procedure: accept the experimenter of percutaneous balloon angioplasty and Stent for each, all accept intubate and with the heart monitor controller, be connected under dorsal position.Introduce 7French sheath pipe through the incision approach to the right carotid entrance, diameter is that the angiopoiesis of 5.0mm marches to femoral artery with sacculus (Guidant Corp. company, Indianapolis, the state of Indiana) under the guide of fluoroscopy.Angiography is completed and record by cineradiography.Expand 30 seconds by sacculus under 8-10 atmospheric pressure, (often stressing futtock 3 times) damaged on right side and left side femoral artery.(Guidant company, 6.0-8.0mm * 18mm) march to femoral artery, and are placed on the position of angioplasty to make the Megalink biliary tract rack under the guide of fluoroscopy.

After angioplasty and Stent Implantation, but comprise endotheliocyte and matrix of microparticles, only comprise matrix of microparticles or the flow composition not to be covered blood vessel orientation on every side that is delivered to left side and right common femoral artery through pumping needle conduit (needle injection catheter) whatever.Adopt angiography for example or intravascular ultrasound art to identify the position of support, with the catheter directed medicine-feeding part of pumping needle.But implantable flow composition is the about position in 1-20mm of mount proximal end for example in the mount proximal end position, and the rack far end position is rack far end approximately in 1-20mm for example, and along the position administration of support total length.Each injection position is all accepted approximately 0.1-1.0ml, comprises approximately 40-70mg microgranule, and density is about 0.8-2.5 * 10 ⁴But the implantable flow composition of individual cell/mg.All experimenters all accept heparin in art, and after operation every day give aspirin.

But 10 experimenters are at the implantable flow composition of operation acceptance on the same day.10 experimenters only accepted to contrast matrix of microparticles the same day in operation.All the other 6 experimenters accept support, but do not accept the implantation of above-mentioned any type.These 6 experimenters are used for comparing with nursing standard.Total cell carrying capacity is approximately 1 * 10 according to body weight ⁴-8 * 10 ⁴Individual cell/kg.

But complete the angiopoiesis program, support is inserted and implantable flow composition after the injection of blood vessels adjacent week gap, C type arm cryptoscope is placed in experimenter's cervical region top, make and be treated angiography.Under continuous fluoroscopy, injection 10-15cc iodine contrast medium (Renograffin, full concentration).Record and store movies angiography, compare with the angiogram before putting to death.Carry out last angiography, estimate the situation of the injection site of vascular patency and implantable microparticle material.

Before angioplasty, give heparin 100U/kg through a notes, and through continuous infusion, give heparin 35U/kg/h and continue to operation to finish.Give in case of necessity extra group's dosage (bolusdose, 100U/kg), to keep ACTs 〉=200 second.

Vascular patency: carry out inlet flow rate with the colorful blood doppler ultrasound immediately after operation and measure,, to determine to be treated the patency rate of blood vessel, carried out once again, and after this carried out once weekly in postoperative 3-7 days.Monitor closely is treated the blood flow of blood vessel.Must detect the flow by blood vessel, until after being placed in experimenter in research and undergoing surgery the 7th day, and comprise the 7th day.If the flow by blood vessel do not detected before the 7th day or the 7th day the time, this experimenter is rejected from this research, and carry out various trials to replace the experimenter, the original amount of experimenter/group is remained unchanged.

The pathology program: the animal subjects to half (is received treatment for 5; 5 contrasts; 3 experimenters do not accept implantation) 3-5 days enforcement euthanasia after operation.Remaining animal subjects (is received treatment for 5; 5 contrasts; 3 experimenters do not accept implantation) implement euthanasia at the hands month after operation.

Animal subjects is anaesthetized with pentobarbital sodium (65mg/kg, intravenous injection).Exposure is treated blood vessel, to being treated blood vessel and surrounding tissue and blood vessel structure, carries out digital camera.Then C type arm cryptoscope is placed in animal's neck, makes and be treated angiography.Under continuous fluoroscopy, injection 10-15cc iodine contrast medium (Renograffin, full concentration).Record cineangiography at 0 ° and 90 ° that is treated blood vessel., by blind reading postmortem angiogram, with the angiogram after inserting, carry out paired comparison and determine vascular patency and narrowness.Angiogram is divided into the 0-5 level according to the narrowness of observing at angiogram.The deciding grade and level scheme that adopts is as follows: 0 grade=0% is narrow, and 1 grade=20% is narrow, and 2 grades=40% is narrow, and 3 grades=60% is narrow, and 4 grades=80% is narrow, and 5 grades=100% is narrow.Expection should show and contrast the experimenter and compare the narrowness that reduces after but the blood vessel of implantable flow composition treatment of the present invention is checking angiogram.

The histology: the animal subjects to half (is received treatment for 5; 5 contrasts; 3 experimenters do not accept implantation) implemented euthanasia in rear 3 days in operation.Remaining animal subjects (is received treatment for 5; 5 contrasts; 3 experimenters do not accept implantation) implement euthanasia at the hands month after operation.

The postmortem that all experimenters all limit, limit postmortem and be defined as macroscopy all experimenter's medicine-feeding part and surrounding tissues, comprises draining lymph node.Implement the experimenter of euthanasia after 1 month for all operations, collect and preserve its major organs and comprise the tissue of brain, lung, kidney, liver, the heart and spleen.Only have when occurring that difference is found in the macroscopy of the macroscopy of body outer surface or medicine-feeding part and surrounding tissue, just organ is analyzed.

All are treated blood vessel and surrounding tissue excision, in stuck-at-１ 0% formalin (or equivalent), and are embedded in glycolmethacrylate (or equivalent).Be cut into approximately 3 μ m slabs with the stainless steel knife of C type (or equivalents), three fragments that are treated blood vessel are taken from the section of preparation: the injection material near-end; The position of injection material; And injection material far-end.These sections are stated from gelatin coating (or equivalent) sheet glass, with haematoxylin and eosin or the dyeing of VerhoeffShi elastin laminin stain.Check the slide after dyeing, the existence that reaches inflammation (acute and chronic), vascular degeneration, thrombosis and fibrosis and smooth muscle cell and the endotheliocyte of lumen of vessels around blood vessel is marked.Other tissue slice available from 1 month experimenter dyes and checks with the VerhoeffShi elastin laminin, and vessel size, blood vessel injury degree, neointimal hyperplasia and restenosis are marked.Other section also can, with specificity endotheliocyte and the dyeing of smooth muscle cell labelling, include but not limited to PECAM-1 and α-SMC actin.Check simultaneously the residual blood tube chamber, with the geometric change of reflection blood vessel after impaired and reparation.Adopt computerized digital planimetry to carry out Morphological Quantitative Analysis with videomicroscopy and customized software the section of Verhoeff Albert'stain Albert again.

The blood vessel of determining acute (experimenters of 3-5 days) and chronic (experimenter of 1 month) reaches the lumen of vessels inflammation on every side.The acutely inflamed granulocyte that is masked as, be mainly neutrophilic granulocyte, and chronic inflammatory disease be masked as macrophage and lymphocyte.In addition, section also can be dyeed with following specific marker: anti-CD45 is used for the identification leukocyte, and anti-CD3 is used for identification T cell, and CD79 is for identification B cell, and MAC387 is used for the identification monocyte/macrophage.

Check the slide after dyeing, the existence of smooth muscle cell and endotheliocyte is marked., to being treated all parts of blood vessel, comprise that middle film outside (adventitia position) and the adventitia of inner, the nearly adventitia of middle film of inner membrance/false inner membrance, hematochezia tube chamber estimated and mark.Measure each take micron as unit and organize for example size of inner membrance, middle film and adventitia of compartment.Each section is estimated with regard to existence and/or the degree of following each standard.The evaluation index of inflammation includes but not limited to neutrophilic granulocyte, lymphocyte, macrophage, eosinophilic granulocyte, giant cell and plasmacytic existence and degree.The existence that fibroblast in evaluation of tissue section, new vessels, calcification, hemorrhage, congested, fibrin, graft fibrosis and graft permeate.In addition tissue slice is carried out the evaluation of degeneration index, include but not limited to degeneration, elastin laminin disappearance and/or tissue part's disappearance, smooth muscle muscle fiber cavity and/or tissue calcification.Also tissue slice is estimated cell proliferation under endothelial cell proliferation, inner membrance and include but not limited to new vessels, and smooth muscle muscle fiber, fibroblast and Fibrotic existence.Each measured tissue slice is also estimated the existence of organizing gangrene and foreign body.Each variable gives by the grade of 0-4 all that minute (0=is without obvious change; 1=is slight; 2=is slight; The 3=moderate; And 4=severe).

To only be stated from from other tissue slice of 1 month animal subjects on sheet glass and dyeing (VerhoeffShi elastin laminin), carry out Morphological Quantitative Analysis.Adopt computerized digital planimetry with videomicroscopy and customized software, volume and total blood vessel volume of the lumen of vessels of each section, middle film, inner membrance to be measured.Measure the narrow percentage rate of each section.A kind of method that quantizes neointimal hyperplasia is with area [(Intimal area, the mm of Intimal area divided by inner membrance and lumen of vessels ²The area of)/(inner membrance+lumen of vessels, mm ²)].

If when rough inspection blood vessel, in the visual observation at above-mentioned position, arrive local damage, blood vessel wall attenuation or diastole, can also obtain other section under pathologist's judgement.Slide after to all dyeing checks and marks in blind (reading) mode by the veterinary pathology person of Professional Certification.

Animal blood vessels gets involved experimenter's expected results

But experimenter's expection for the treatment of through flow composition of the present invention as described above demonstrates one or more blood vessels and gets involved the index that the occurrence frequency of relevant clinical sequela reduces, and includes but not limited to that occluding thrombus minimizing, patency rate increase, narrow minimizing, neointimal hyperplasia reduce and acute and chronic vascular chamber and/or the minimizing of periangiitis disease.

The index of another Successful treatment blood vessel is enough lumen of vessels diameters.Estimate that thereby injectable materials of the present invention allows the unimpeded blood flow of normal or nearly normal speed to safeguard enough lumen of vessels diameters by reducing angiostenosis.Before same day and facing being condemned to death on the 30th day, treatment is treated lumen of vessels diameter and the narrow percentage rate of blood vessel with the angiography monitoring.With standard doppler ultrasound scheme, postoperative the narrowing down of lumen of vessels connected with rate of blood flow.Expection the present invention can prevent or postpone to narrow down, thereby stop rate of blood flow lower than normal or near normal speed.

Another index that functional support is inserted blood vessel is the disappearance of edge effect.Injectable materials of the present invention can reduce rack far end and the occluding thrombus disease of mount proximal end vasculature part or narrow generation and/or the degree (so-called edge effect) that is treated blood vessel.Edge effect or candy wrapper effect refer to support insert after in the zone of support junction (articulations) and the narrow development in edge.Edge effect is narrow in feature take early stage new intima hyperblastosis and late period, can cause occluding thrombus disease.Before treatment was condemned to death at same day and facing on the 30th day, with angiography, the edge effect that is treated blood vessel is monitored.Relevant thrombosis and the blood vessel of edge effect that the present invention can prevent as described herein like that or delay and support are inserted blood vessel narrows down.The existence of edge effect or disappearance also can be by obtaining utmost point near-end and the section of utmost point far-end, i.e. the two extrorse upstreams of support or the approximately section at 2-3mm place of downstream, and it is carried out histological examination determine.For the existence of evaluation edge effect, damage, inflammation, Neointimal formation and the thrombosis of utmost point near-end and the section of utmost point far-end are marked.

Being treated subject group compares with matched group and has at least a kind of aforementioned functional index to demonstrate the difference of increment type at least.

Embodiment 2: people's blood vessel Intervention studies

But the present embodiment provides in the clinical experimenter of people test and has used the vascular endothelial cell that comprises transplanting and the flow composition of the biocompatible matrix of particulate form, with the testing program of the generation that reduces the clinical complication relevant with the blood vessel intervention.Adopt SOP, complete percutaneous balloon angioplasty and the stenting controlled by the doctor, to alleviate clinical disease.Then but implantable flow composition is deposited on the perivascular canal at angioplasty and stenting position, perhaps with the adjacent perivascular canal of angioplasty and stenting position, perhaps near the perivascular canal angioplasty and stenting position; The details of an exemplary program is set forth hereinafter.As previously mentioned, but inserting with preparation of implantable flow composition can be changed in the usual way by skilled practitioner.

Especially, this research is included in periphery limbs (peripheral limb) and accepts the people experimenter of percutaneous balloon angioplasty and stenting.Carry out traditional percutaneous balloon angioplasty and stenting operation according to the standard operation technology.Complete angioplasty and stenting, and setting up by after the blood flow that is treated blood vessel, but implantable flow composition of the present invention is being applied to the position of balloon expandable and on every side as described above.Total cell carrying capacity is approximately 1.0 * 10 based on body weight ⁴Individual cell/kg is to approximately 8 * 10 ⁴Individual cell/kg.

Clinical Follow-up the 5th day, 2 the week and carried out in 1,3,6 month.Carried out blood flow measurement to set up baseline values with the colorful blood doppler ultrasound in the 5th day after surgery, then 2 weeks, 1 month, 3 months and 6 months are carried out blood flow measurement after surgery.To showing absolute flow rate less than 350mL/min, or blood flow reduces greater than 25% than previous measured value, or narrow area is greater than experimenter's promoting the circulation of blood pipe visualization of 50% (with doppler ultrasound, measuring).Permission determines that to the intravascular radiography Stenotic pathologic change carries out for example angioplasty of the clinical intervention of therapeutic greater than 50% experimenter.

Compare angiography baseline day and 3rd month to being treated blood vessel and surrounding tissue and blood vessel structure.Calculate each regional lumen of vessels diameter, measure peak systolic velocity.

The result of people's blood vessel Intervention studies

As described above, but the experimenter through flow composition treatment of the present invention demonstrates one or more blood vessels intervention relevant clinical sequela, includes but not limited to the index of the occurrence frequency minimizing of occluding thrombus, restenosis, neointimal hyperplasia and acute and chronic inflammation.

An index of functional blood vessel is enough lumen of vessels diameters.The present invention can allow to keep enough lumen of vessels diameters, thereby permissible velocity is enough to keep the unimpeded blood flow of normal or near normal circumference circulation.First, in baseline day (rear approximately 5 days for the treatment of), then with angiography, the lumen of vessels diameter that is treated blood vessel was monitored at least 3 months after surgery.With standard doppler ultrasound scheme, postoperative the narrowing down of lumen of vessels connected with rate of blood flow.Can prevent or postpone when but implantable flow composition of the present invention uses by methods described herein to narrow down, thereby stop rate of blood flow lower than the speed that is suitable for peripheral circulation described herein.Further, but with implantable flow composition treatment of the present invention, can produce the permission rate of blood flow of acceptable circulation clinically, or near the rate of blood flow of normal speed.It is comparable flowing into or flowing out the blood flow that is treated the part blood vessel.The comparable meaning and clinical application broadly similar.For example, rate of blood flow is about 150-500mL/min, preferred approximately 300-500mL/min, more preferably from about 350-400mL/min.

Being treated subject group compares with matched group and has at least a kind of aforementioned functional index to demonstrate the difference of increment type at least.

Embodiment 3: the adhesion research again of animal pelvis

But the present embodiment provides to test in animal subjects and use and has comprised the endotheliocyte of biocompatibility matrix of microparticles and transplanting or the flow composition of endothelioid cells, the experimental program that occurs to reduce pelvis and adhesion on every side thereof.Adopt rat experiment model (the cornua uteri model of improvement, J.Invest.Surg.7:409-15 (1994)) but research is treated the reconstruction of fallopian tube posterior synechiae with implantable flow composition of the present invention and method.Scraping cornua uteri both sides and with its stitching.After 14 days, the tight junction between the cornua uteri both sides is sewed up in excision in abdominal part is performed the operation again.As described above but implantable flow composition of the present invention is applied to cornua uteri one side and around.But the opposite side of cornua uteri is not accepted implantable flow composition, in contrast.Constantly monitor existence or the disappearance of adhesion.But the rat through implantable flow composition treatment of the present invention demonstrates reducing of pelvis and adhesion on every side thereof.

Embodiment 4: the research of animal tubal occlusion

The present embodiment provides in animal subjects test and has used endothelium or the endothelioid cells of biocompatible matrix and transplanting, with the experimental program of the generation that reduces tubal occlusion.Adopt rabbit experimental model (J.Vase.Interv.Radiol.13:399-404 (2002)) but research use implantable flow composition of the present invention and method treatment tubal occlusion.Under the guide of fluoroscopy mirror, adopt coaxial technology in right side and the left side fallopian tube vagina catheterization of passing through.Stretch out wire guide from the active electrode conduit, carry out the RF electric coagulation.As described above but implantable flow composition of the present invention is applied to an oviductus lateralis and around.But the opposite side fallopian tube is not accepted implantable flow composition, in contrast.Constantly monitoring tubal patency rate and histology change.But the rabbit through implantable flow composition treatment of the present invention demonstrates fallopian tube and inaccessible, narrow and downright bad reducing on every side.

The present invention also can be used for effectively reducing the generation of ectopic pregnancy and/or as ectopic pregnancy simultaneously or interventional therapy afterwards.

The present invention can embody with other specific form under the condition that does not exceed its spirit or substitutive characteristics.Therefore current embodiment is interpreted as illustrative and nonrestrictive, scope of the present invention is limited by the description of claim rather than front, and all implications in the equivalents of this claim and the variation in scope, all therefore will comprise in the present invention.

Claims (42)

1. treat the flowable compositions of the impaired or disease sites of tubular anatomical structures inner chamber, wherein said flowable compositions comprises biocompatible matrix and endotheliocyte, wherein said endotheliocyte is paved with, that closely be paved with or be paved with after and be implanted on described biocompatible matrix, among and/or within, and described biocompatible matrix comprises microgranule or microcarrier, and described microgranule or microcarrier further comprise gelatin, collagen protein, fibronectin, fibrin, laminin，LN, or be selected from the attaching peptide of the peptide of sequence arginine-Gly-Asp (RGD), but described flow composition is delivered to impaired by percutaneous injection or disease sites or near chamber outer surface adjacent with this position or this position, and the amount of described flowable compositions is the amount of impaired or disease sites of effectively treating.

2. the flowable compositions of claim 1, wherein the flowable compositions is to keep shape.

3. the flowable compositions of claim 1, the diameter of wherein said microcarrier is 20 microns to 500 microns.

4. the flowable compositions of claim 1, the diameter of wherein said microcarrier is 200 microns.

5. the flowable compositions of claim 1, it further comprises suitable carrier fluid, described carrier fluid is selected from alginate, sugar juice, starch solution, gelatin, endothelial cell growth culture medium, endothelial basal medium or neutral buffered salt.

6. the flowable compositions of claim 1, wherein said effective dose reduces the propagation of the smooth muscle cell of impaired or disease sites.

7. the flowable compositions of claim 1, wherein said effective dose alleviates the occluding thrombus of impaired or disease sites.

8. the flowable compositions of claim 1, wherein said effective dose reduces the neointimal hyperplasia of impaired or disease sites.

9. the flowable compositions of claim 1, wherein said effective dose alleviates the narrow or restenosis of impaired or disease sites.

10. the flowable compositions of claim 1, wherein said effective dose alleviates the acute inflammation of impaired or disease sites.

11. the flowable compositions of claim 1, wherein said effective dose alleviates the chronic inflammatory disease of impaired or disease sites.

12. the flowable compositions of claim 1, wherein said effective dose alleviate vasodilation or the vasospasm of impaired or disease sites.

13. the flowable compositions of claim 1, wherein said tubular anatomical structures is blood vessel.

14. the flowable compositions of claim 1, wherein said compositions is sent by minimally-invasive closed operation method.

15. the flowable compositions of claim 1, wherein said compositions are to send by the inwall that passes or see through described tubular anatomical structures.

16. the flowable compositions of claim 1, wherein said tubular anatomical structures are the anastomotic stoma that natural structure or operation produce.

17. but biocompatible matrix and the endotheliocyte purposes in the flow composition of the impaired or disease sites for the preparation for the treatment of tubular anatomical structures inner chamber, wherein said endotheliocyte is paved with, that closely be paved with or be paved with after and be implanted on described biocompatible matrix, among and/or within, and described biocompatible matrix comprises microgranule or microcarrier, and described microgranule or microcarrier further comprise gelatin, collagen protein, fibronectin, fibrin, laminin，LN, or be selected from the attaching peptide of the peptide of sequence arginine-Gly-Asp (RGD), but wherein said flow composition is delivered to following site by percutaneous injection and described flowable compositions is contacted with this site: the exocoel surface of the impaired or disease sites of described tubular anatomical structures or or near this position described tubular anatomical structures adjacent with this position, and the amount of wherein said flowable compositions is the amount of impaired or disease sites of effectively treating.

18. the purposes of claim 17,, wherein by passing or see through the inwall of described tubular anatomical structures, be deposited on the flowable compositions impaired or disease sites or adjacent with this position or near the outer surface of the described tubular anatomical structures this position.

19. the purposes of claim 18, wherein identify the flowable compositions deposits on the outer surface of described tubular anatomical structures position.

20. the purposes of claim 19, wherein before passing or see through the inwall of described tubular anatomical structures or identify at the same time the deposition site of described flowable compositions.

21. the purposes of claim 19, wherein identify described position by imaging.

22. the purposes of claim 19, wherein identify described position by palpation.

23. the purposes of claim 17,, wherein by percutaneous dosing intravasation week gap, be deposited on the flowable compositions impaired or disease sites or adjacent with this position or near the outer surface of the described tubular anatomical structures this position.

24. the purposes of claim 23, wherein identify the flowable compositions deposits on the outer surface of described tubular anatomical structures position.

25. the purposes of claim 24, wherein by before percutaneous dosing intravasation week gap or identify at the same time the deposition site of described flowable compositions.

26. the purposes of claim 24, wherein identify described position by imaging.

27. the purposes of claim 24, wherein identify described position by palpation.

28. the purposes of claim 17, the outer surface of wherein said tubular anatomical structures are surfaces, non-chamber.

29. the purposes of claim 17, the outer surface of wherein said tubular anatomical structures has occupied perivascular canal.

30. the purposes of claim 17, wherein said tubular anatomical structures is blood vessel.

31. the purposes of claim 30, wherein said blood vessel comprises support.

32. the purposes of claim 31, wherein said impaired or disease sites is near support.

33. the purposes of claim 17, but the described effective dose of wherein said flow composition reduces the propagation of the smooth muscle cell of impaired or disease sites.

34. the purposes of claim 17, but the described effective dose of wherein said flow composition alleviates the occluding thrombus of impaired or disease sites.

35. the purposes of claim 17, but the described effective dose of wherein said flow composition alleviates the neointimal hyperplasia of impaired or disease sites.

36. the purposes of claim 17, but the described effective dose of wherein said flow composition alleviates the narrow or restenosis of impaired or disease sites.

37. the purposes of claim 17, but the described effective dose of wherein said flow composition alleviates the acute inflammation of impaired or disease sites.

38. the purposes of claim 17, but the described effective dose of wherein said flow composition alleviates the chronic inflammatory disease of impaired or disease sites.

39. the purposes of claim 17, but the described effective dose of wherein said flow composition alleviates vasodilation or the vasospasm of impaired or disease sites.

40. the purposes of claim 17, wherein said tubular anatomical structures is selected from: tear stains, trachea, bronchus, bronchioles, nasal meatus, nasal sinuses, pharyngotympanic tube, external auditory meatus, oral cavity, esophagus, Stomach duodenum, small intestinal, large intestine, biliary tract, ureter, bladder, urethra, fallopian tube, uterus, vagina and deferent duct.

41. the flowable compositions of the impaired or disease sites for the treatment of tubular anatomical structures inner chamber, wherein said flowable compositions comprises biocompatible matrix and endotheliocyte and suitable carrier fluid, and the amount of described flowable compositions is the amount of impaired or disease sites of effectively treating, wherein said endotheliocyte is paved with, that closely be paved with or be paved with after and be implanted on described biocompatible matrix, among and/or within, wherein said biocompatible matrix comprises microgranule or microcarrier, and described microgranule or microcarrier further comprise gelatin, collagen protein, fibronectin, fibrin, laminin，LN, or be selected from the attaching peptide of the peptide of sequence arginine-Gly-Asp (RGD).

42. the flowable compositions of claim 41, wherein said carrier fluid are selected from alginate, sugar juice, starch solution, gelatin, endothelial cell growth culture medium, endothelial basal medium or neutral buffered salt.

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