CN101076360A

CN101076360A - Materials and methods for minimally-invasive administration of a cell-containing flowable composition

Info

Publication number: CN101076360A
Application number: CNA200580042372XA
Authority: CN
Inventors: 海伦·玛丽·纽金特; 埃拉泽尔·埃德尔曼; 史蒂夫·博林格
Original assignee: Pervasis Therapeutics Inc
Current assignee: Pervasis Therapeutics Inc
Priority date: 2004-12-08
Filing date: 2005-12-06
Publication date: 2007-11-21
Anticipated expiration: 2025-12-06
Also published as: ES2348961T3; CN101076360B; CN101141988A; CN101141988B; CN101166484A

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 material of the minimally-invasive administration of celliferous flow composition and method

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 submitted on May 19th, 2005,054, and _ _ _ _ priority of the temporary patent application U.S.S.N.60/ that submits to; And according to United States code 35 chapters and sections 120,363 and/or 365, require co-pending International Patent Application PCT/US_____ (being also referred to as the case ELV-002PC of agency) of submitting on the same day with the application, and the priority of the co-pending International Patent Application PCT/US_____ (being also referred to as the case ELV-008PC of agency) that submits on the same day with the application; 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 above 500,000,000 people's death every year, causes 1 million people's death in the U.S..In the U.S., approximately finish 1,500,000 operations every year to attempt to alleviate the obstructive arterial disease that causes because of these idiogenesis.Several sacculus angioplasty and laser angioplasty, speckle rotary-cut art, endovascular stent art and bypass graft etc. of comprising of these operations that can mention.The long-term effect of angioplasty, blood vessel by-pass grafting even transplant operation is 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 typical case's representative of this class arteriopathy.For example, restenosis causes the 20-50% among these patients that the obstructive arterial disease takes place.In 3-6 behind coronary angioplasty month, surpass 1/3rd extra interventional procedure, another time angioplasty even the by-pass operation 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 takes place 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, except that acute complications takes place about 5% patient, outside closing suddenly, the ratio that restenosis takes place is 35%.

In accepting the patient of vascular bypass operation, observed similar problem.The average life that saphena aorta-coronary artery inserts transplantation only is 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 incident 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 works as the physiological dual bioregulator of blood vessel.Endothelium provides the integrity of structure by form successive, the permeable non-thrombinogen barrier of selectivity between blood circulation and arterial wall 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 the arterial wall, is that the existence of complete endothelium makes normal blood vessels maintain resting state.In vessel endothelium and induced endothelial or after removing, be removed similarly by the excretory chemical compound of endothelium, and begin to take place a series of incidents, 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 cardiovascular disease at present comprises coronary angioplasty, intracoronary stening and speckle rotary-cut art, can adopt Noninvasive closed operation method to finish.The interventional procedure strategy should be got involved the position to treat the intravascular modalities of therefore impaired or ill target endothelium with the delivery of therapeutic material that has minimally-invasive equally to blood vessel in these Noninvasive blood vessels.

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 limited active effectiveness and persistent period, bestow the therapeutic material or reagent only can provide temporary transient effect to impaired or ill target endothelium to surface of internal cavity with contacting of blood circulation.

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

Summary of the invention

The present invention has utilized such discovery: but transplant among the 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 near this outer surface, carry out depositing around intravascular administration and the blood vessel, described anatomical structure is such as but not limited to blood vessel.At the outer surface of tubular anatomical structures, or on the outer surface, or the minimally-invasive of external surface peripheral is sent and is also included among the present invention.Under the situation of blood vessel, material of the present invention is suitable for treating with control with method to be got involved or the relevant clinical sequela of cardiovascular disease with blood vessel.

On the one hand, but the present invention is the flow composition of the impaired or disease sites of treatment 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 a blood vessel.According to some embodiments, but flow composition is impaired with effective minimizing or for example smooth muscle cell proliferation of disease sites, occluding thrombus disease, neointimal hyperplasia, restenosis, amount acute or chronic inflammatory disease or vasodilation etc. provide.For purpose of the present invention, but be meant can be with the compositions of injecting or 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 among 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.Be applicable to that 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 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 about 20 microns to about 500 microns, and preferred diameter is about 200 microns.

In another embodiment, but flow composition further comprises carrier fluid.In especially preferred embodiment, remain unchanged but flow composition is a shape, thereby the permission practitioner controls the deposition to specified particular deposition position on the degree of necessity.

On the other hand, the present invention is the method for the impaired or disease sites of treatment tubular anatomical structures inner chamber.This 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 (being also referred to as outside the chamber) herein can be the outer surface or the circumvascular surface of blood vessel.For purpose of the present invention, the position is any position except the inner surface in chamber outside non-chamber or the chamber.Under the situation of blood vessel, for example, outside the chamber or exocoel position can be in the adventitia of blood vessel, middle film or inner membrance; Under the situation of 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, be positioned at impaired or disease sites then, or adjacent with this position, but or near the outside deposition flow composition of the tubular anatomical structures this position finish and send.According to another embodiment, but this method further is included in the step that the chamber outer surface of tubular anatomical structures is identified the position of deposition flow composition.According to an embodiment, this authentication step occurs in this and passes or see through before the step, perhaps passes or sees through step with this and take place simultaneously.In one embodiment, this authentication step is finished by imaging.In another embodiment, this authentication step is finished by palpation.

In one embodiment, by through percutaneous dosing intravasation week crack, 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 finish and send.According to another embodiment, but this method further is included in the step that the outer surface of tubular anatomical structures is identified the position of deposition flow composition.This authentication step can occur in this and enter before the step, perhaps enters step with this and takes place simultaneously.According to an embodiment, this authentication step is finished by imaging.In another embodiment, this authentication step is finished by palpation.

According to the various embodiments of this method, the outer surface right and wrong chamber of tubular anatomical structures surface or occupy at the described perivascular canal in this paper other places.According to a preferred embodiment, tubular anatomical structures is a blood vessel.According to another preferred embodiment, blood vessel comprises support.In a further preferred embodiment, the tubular anatomical structures of being 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 explained, 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 are got involved or cardiovascular disease relevant clinical sequela, especially occluding thrombus disease, restenosis, neointimal hyperplasia, inflammation and vasodilative progress.The present invention further benefits from another discovery, promptly, but still unrecorded up to now flow composition, microparticle formulation for example, can provide the vigor that is paved with competent cell mass, said composition comprise transplant among for example implantable microparticle material of biocompatible matrix, 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 listing below is to manufacturing and use material of the present invention and method that sufficient guidance is provided, and the standard and the experimenter that further the performance of material of the present invention and method are tested, measured and monitors in definite suitable being used to provide sufficient guidance.

Therefore, developed the therapy that is used for controlling clinically blood vessel intervention or cardiovascular disease based on cell.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 endotheliocyte and particle type biocompatible matrix.

But implantable flow composition of the present invention comprise transplant on the biocompatible matrix, among and/or within cell.The meaning of transplanting is meant through cell pair cell and/or cell adheres 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 the back cell mass of preferred phenotype.Natural is, in preparation property operating period, but exfoliative cyte and/or some cell do not resemble and adhere to securely other cell the embodiment of implantable flow composition probably.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 satisfies above-mentioned clinical demand.But the unique distinction of implantable flow composition of the present invention is, transplant on the biocompatible matrix, among and/or within living cells can under the physiology feedback control, provide the product that is in the physiology ratio based on various kinds of cell to tubular anatomical structures.As described elsewhere herein, but be applicable to that the cell of implantable flow composition is endotheliocyte or endothelioid cells.The local delivery of the multiple chemical compound 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 the endotheliocyte in the implantable flow composition for example of the present invention has been avoided the destruction of the aggressivity blood flow of intravascular space owing to it preferably places position outside non-chamber or the chamber.

But when implantable flow composition of the present invention with parcel, deposition or alternate manner and be positioned at impaired or ill target chamber, or adjacent, or during the contact of outside near the chamber this target chamber or position, non-chamber, it is used to rebuild homeostasis with this target chamber.Promptly when the external administration of chamber, simulate supportive physiological environment, and help lend some impetus to the growth of functional inner chamber but implantable flow composition of the present invention can provide.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 an 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 the 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 with 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 deposit implantable flow composition of the present invention at the perivascular canal of outside, target blood structure chamber.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 chemical compound.Though 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 the 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, under situation about lacking 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 from body endotheliocyte and smooth muscle cell to be exceedingly fast or velocity anomaly growth out of control.Therefore, as this paper expection like that, but adopt the treatment of implantable flow composition of the present invention will produce that blood vessel is got involved or the normal or closely normally recovery of the autologous tissue at 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 place by the treatment 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 be meant directly or indirectly as with defined chamber, this paper other places outside or non-chamber surface interaction.Under the situation of some preferred embodiment, the actual physical contact is not that effectiveness is necessary.In other embodiments, the 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.Under the situation of some i or I, ill or damaged part can find expression in surface of internal cavity clinically.Under the situation of other i or I, ill or damaged part can find expression in outside the chamber or surface, non-chamber clinically.In some diseases or damage, ill or damaged part can find expression in outside surface of internal cavity and the chamber or surface, 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 the 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 an 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 with the blood vessel that causes blood vessel injury of the present invention's treatment and include but not limited to angioplasty, speckle rotary-cut art, intravascular stent (comprising naked metal rack 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 are migrated to the shape art, and the angioaccess associated injury that takes place 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 that occluding thrombus disease, restenosis, neointimal hyperplasia, acute and chronic inflammation, smooth muscle cell proliferation, blood vessel are reinvented, the formation of vasodilation and vulnerable plaque pathological changes.

Thrombosis or occluding thrombus disease are relevant with hematoblastic adhesion, gathering and machineization; Occluding thrombus disease is relevant with organized thrombus usually.Thrombosis is characterised in that the blood flow disappearance in the 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 can improve by the patency rate of treatment blood vessel with implantable flow composition treatment of the present invention.

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 chemical 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 the narrow percentage rate of implantable flow composition treatment can reducing of the present invention, inaccessible degree and/or raising and by the relevant patency rate of treatment 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 increases the speed that the phagocyte migration is passed the surface, chamber and entered adjacent tissue greatly.After the activation, these cells can discharge hydrolytic enzyme, cytokine, chemotactic factor and somatomedin.In the chronic progressive stage of inflammation, damaged part becomes fibrous cap bag quilt, and this fibrous cap covers on fat nuclear and the slough.Endothelium or endothelioid cells discharge the anti-inflammatory compound that can reduce inflammatory reaction in cavity region.But can the inflammation-inhibiting cell activity and/or gather with implantable flow composition treatment of the present invention, therefore the 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 incident 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.Further discussion to angiopathy is disclosed among the co-pending application PCT/US_________ (the case ELV-008PC of agency) that submits on the same day with the application, its full content is incorporated herein by reference, 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: acute and chronic inflammation, occluding thrombus disease, neointimal hyperplasia, restenosis, smooth muscle cell proliferation, vasodilation, negativity blood vessel are reinvented, the vulnerable plaque pathological changes of blood vessel structure intracavity and various unstability superior mesenteric artery syndromes 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 influenced the damage of blood supply or be repaired and produce by any negativity.Exemplary rapid wear disease comprises the unstability superior mesenteric artery syndrome, for example 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, the first time blood vessel get involved, but for example adopt the impaired or ill target endothelium of implantable flow composition treatment of the present invention angioplasty, stent endoprosthesis or anastomosis the time.This treatment can reduce to get involved the damage cause by blood vessel, 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 with 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 extra therapeutic agent afterwards.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 in implantable flow composition, add other cytokine or somatomedin, 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 the implantable flow composition.

The cell source: such as described herein, but implantable flow composition of the present invention comprises cell.Cell can be allochthonous, xenogeneic 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 a present embodiment preferred, cell is an 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 suitable vascular endothelial cell type is an aortic endothelial cell.The another kind of vascular endothelial cell type that is suitable for is a huve cell.Another vascular endothelial cell type that is suitable for is a coronary artery endothelial cell.Other is applicable to that vascular endothelial cell type of the present invention comprises pulmonary artery endothelial cell and iliac endothelial cells.

In another present embodiment preferred, 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, first kind of cell imported in the implantable material of biocompatibility, and be cultured to and be paved with.First kind of 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 first kind of cell type reaches the state of being paved with, then second kind of cell type is seeded in be positioned among the implantable material of biocompatibility, on or within first kind be paved with on the cell type, and be cultured to first kind of cell type and second kind of cell type all reaches the state of being paved with.Second kind of 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 import as single mixture.Also can change the density of cell, thereby transplant for AV smooth muscle cell be become about 2: 1 to the ratio of endotheliocyte, for the periphery coronary artery bypass grafting ratio be become about 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 first kind of cell type is paved with, before importing second kind of cell type, can earlier culture be covered with the attachment element that is suitable for second kind of cell type.Exemplary attachment element comprises with gelatin and covers culture, to improve the tack of endotheliocyte.According to another embodiment, between second kind of cell type culture period, in culture medium, add the propagation that heparin can reduce by first kind of cell type, and optimize the first kind of cell type of needs and the ratio of second kind of cell type.For example, after the smooth muscle cell initial growth, can give the growth of heparin, thereby reach bigger endotheliocyte ratio smooth muscle cell with the control 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, then endotheliocyte is inoculated on the smooth muscle cell of cultivating on the implantable material, to produce simulated blood vessel.This embodiment can be applied to for example AV transplanting or periphery bypass graft according to methods described herein, 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 (describing in this paper other places) associating, and it can promote, repair and/or otherwise regulate and the relevant endotheliocyte physiology and/or the intracavity ambient stable in impaired or ill target chamber for the treatment of impaired or ill target vessel endothelium or another tubular anatomical structures.

For purpose of the present invention, a kind of preferred typical cells phenotype of easily identifying of the present invention is by analyzed in vitro method measurement hereinafter described, 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 platelet adhesion and accumulative phenotype.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 show is no more than a kind of in the aforementioned phenotype.In certain embodiments, cell can show more than one aforementioned phenotypes.

Though aforementioned phenotype is represented 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, be applicable to that the cell of material disclosed herein and method 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 during with the associating of as described herein those of preferred biocompatible matrix, 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 the back cell mass can easily identify by multiple technologies, the extensive recognized techniques of wherein the most frequently used and quilt 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, on function and phenotype, imitate or simulate the cell that closely is paved with, is paved with and be paved with the back endotheliocyte.

Therefore, utilize hereinafter described to 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.At last, all essential conditions are, can effectively treat, control, regulate or alleviate chamber damage or disease according to these equivalents of method disclosed herein, for example damage or disease with chamber that blood vessel is got involved or cardiovascular disease is relevant as non-limitative example.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 freezing preservation of known technology and stock, is cultivated expansion after treating, make implantable compositions subsequently.

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 endotheliocyte separation of implantable flow composition from cadaveric donors.In other embodiments, (Cell Applications, San Diego is CA) by separating from the normal pig aorta with the similar program of separation of human aortic endothelial cell for the porcine aorta endotheliocyte.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 the implantable compositions of biocompatibility subsequently.

People or porcine aorta endotheliocyte prepare in the pretreated T-75 bottle of every bottle of about 15ml of adding endothelial cell growth medium.The human aorta endotheliocyte is preparation in endothelial cell growth culture medium (Endothelial Growth Media, EGM-2, Cambrex Biosciences company, EastRutherford, New Jersey).EGM-2 is made up 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 couveuse, at about 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 the refrigerator from-160 ℃ to-140 ℃, under about 37 ℃, thaw.Every bottle of cell after thawing is with preferred about 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 will contain cell is put back in the couveuse then.After about 8-24 hour, remove culture medium (spent media), and replace with fresh culture.After this, changed a subculture in every 2-3 days, reach preferred about 85-100%, but be not less than 60% and be not more than 100% degree of being paved with until cell.When but implantable flow composition is intended for use when clinical, but only cultivate and the production of implantable flow composition of the present invention antibiotic-free culture medium the thawing back that can be used for the human aorta endotheliocyte.

Remove the endothelial cell growth culture medium then, cell monolayer washes 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 5ml HEPES in addition, cell is counted with hematimeter.Cell suspending liquid is centrifugal, under the situation of people's cell, regulate density to about 1.75 * 10 with antibiotic-free EGM-2 ⁶Individual cell/ml, or under the situation of pig cell, regulate density to about 1.50 * 10 with the EBM-2 that is added with 5% FBS and 50 μ g/ml gentamycins 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 the cell growth and be attached to substrate, thereon or in the substrate.When implanting the outer surface of blood vessel for example, biocompatible matrix can rest on the about 7-90 of implant site days 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 be meant can with inject or 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 among the present invention.Any biocompatible matrix that is used for the non-solid form of injection type delivery apparatus includes in the present invention, and device wherein can be implemented intravascular administration or local percutaneous dosing by advancing along internal blood vessel length.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 to 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 pin of 1 to 20mm.The preferred about 50mg of injection delivery apparatus transmissibility, at the about 1 implantable microparticle material that contains 100 ten thousand cells of having an appointment to 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) (to call " Gelfoam microgranule " in the following text).According to another embodiment, the biocompatibility microparticle material is Cytodex-3 (AmershamBiosciences company, Piscataway, a New Jersey) microcarrier, and it is by forming with the bonded denatured collagen of cross-linking dextran substrate.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, Polyethylene Glycol 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 of treatment 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, the function of cell when combining with implantable matrix of microparticles with optimization and/or control cell, described cell comprises the phenotype (for example, inhibition phenotype) of cell mentioned above.According to an embodiment; improvement to implantable matrix of microparticles comprises that with attachment element or adhesin polypeptide coated particle this attachment element or adhesin polypeptide can strengthen cell to be suppressed the generation of smooth muscle cell proliferation, minimizing inflammation, the generation that increases Heparan sulfate, increase 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 having the peptide that cell adhesion is identified 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 that 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 having the peptide that cell adhesion is identified 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 strengthening its mechanical property, and promote its cell attachment and growth characteristics.According to preferred embodiment, it is crosslinked that the alginate microgranule earlier carries out the first time with calcium sulfate, and it is crosslinked to carry out the second time with calcium chloride then, and scheme is carried out routinely at last.According to another embodiment, with the source of heparin or Heparan sulfate, for example heparin-agarose adds in the substrate before cell culture.

But the implantable flow composition that comprises the biocompatibility matrix of microparticles is that No. 22 standard size to No. 26 standard-sized pins are sent with internal diameter preferably.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 extremely about 1000 μ m of about 20 μ m, and preferred diameter is extremely about 500 μ m of about 100 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 can adhere on each microgranule 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 Gelfoam microgranule of about 50-60mg is placed in the single 50mL culture tube (Evergreen company, Los Angeles, California) that has 0.2 μ m filter cap.For reducing the particle number that culture medium was lost between the stage of replacement, can improve culture tube, for example,, when removing culture medium, be discharged from or other is unpredictably removed with the prevention microgranule at the medicated cap district or the inner filter membrane that adds of culture tube; Adding in culture tube can be by culture medium but can not be by the barrier film of microgranule, with form between each several part by the fluid connection isolating culture medium partly and the microgranule part; Perhaps add and not upset the nozzle of opening and emitting culture medium under the particulate condition in the culture tube bottom.

Cell inoculation is gone into implantable material: before cell inoculation, add 70% ethanol in microgranule, then with PBS or HEPES flushing several.Microgranule is in antibiotic-free EGM-2, at about 37 ℃, 5% CO then ₂Under/95% conditions of air rehydrated 12 to 24 hours.Aliquot that then will about 50-60mg microgranule is taken out from its rehydrated container, places 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 down for several times, to form the suspension of homogeneous.Culture tube is at 37 ℃, 5% CO then ₂, 90% humidity condition under regularly stir and hatched 3-4 hour, to promote cell attachment.Add other 9mL EGM-2 (final culture volume is 10mL) subsequently in every pipe, obtaining final microgranule volume ratio is about 5mg microgranule/mL culture medium.

After this, changed a subculture, and reached the state of being paved with in every 2-3 days until cell.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 analysis, with the evaluation cell growth characteristics, and determine whether to reach closely be paved with, be paved with or be paved with after.But the preparation that contains the implantable flow composition of porcine aorta transplanted endothelial cell thing, its representational growth curve is seen Fig. 1.In this embodiment, the form of implantable material is a microgranule.Normally, those of ordinary skills understand acceptable cell growth indexes 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, be closely to be paved with phase (for Fig. 1, being about 10-13 days) then, then be cell reach be paved with the back cell number stable phase (for Fig. 1, be about 13-15 days) and cell be paved with back the keeping the phase (for Fig. 1, being about 15-17 days) of cell number.For purpose of the present invention, preferred at least 72 hours cell mass in stable phase.

But cell counting by with implantable flow composition aliquot with the solution (be applicable to Gelfoam microgranule) of 0.8mg/ml Collagenase in insulin-EDTA, perhaps the solution of glucanase and insulin-EDTA (being applicable to the Cytodex-3 microgranule) decomposes fully and finishes.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 measures survival rate with the trypan blue exclusion method.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.Reach be paved with after, 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 about 8 * 10 at least ³There is total cell number about 7 * 10 in individual cell in preferred every aliquot 50-70mg microgranule ⁵To about 1 * 10 ⁶Individual, and its survival rate is preferably at least about 90% but be not less than about 80%.Cell survival rate preferably is at least about 90% but be not less than about 80%.If cell was not paved with after 12 or 13 days, then change culture medium, continued to hatch one day.This process of continuing is paved with until reaching, perhaps until inoculating about 14 days of back.If determine that after the process inspection cell is paved with, then carry out last culture medium and change.Last culture medium is changed with no phenol red and EGM-2 antibiotic-free and is carried out.After culture medium is changed, immediately culture tube is set up the asepsis plug sealing cap and be used for transportation.

Functional evaluation: 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 is with Heparan sulfate, the transforming growth factor of determining to be produced by endothelial cells cultured 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 to equal 90%, and most preferably at least about 90% o'clock, but implantable flow composition can be used for purpose described herein.Heparan sulfate in the conditioned medium is at least about 0.5-1.0 μ g/10 ⁶Individual cell/sky is preferably at least about 1.0 μ g/10 ⁶Individual cell/sky.TGF-β in the conditioned medium ₁Be at least about 200-300picog/ml/ days, preferably at least about 300picog/ml/ days; B-FGF in the conditioned medium is lower than about 200picog/ml, preferably is no more than about 400picog/ml.

The level of Heparan sulfate can adopt conventional dimethylated methylene base indigo plant-chondroitin sulfate lyases ABC digestion photometry to carry out quantitatively.Total sulphation glycosaminoglycans (GAG) level is measured with dimethylated methylene base indigo plant (DMB) dye binding method, this method compares unknown sample and standard curve then by obtaining standard curve with the aching and limp ossein of bright sulfur of collecting culture medium (collection media) dilution known quantity.Before adding the DMB developer, other sample of conditioned medium is mixed with chondroitin sulfate lyases ABC, with decomposition 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 about 515-525nm and measure.By deduct the concentration of chrondroitin and dermatan sulfate with the total sulphation glycosaminoglycans concentration in the conditioned medium sample, calculate per 10 ⁶The Heparan sulfate level of individual cell every day.The activity of chondroitin sulfate lyases 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%, then 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 in the presence of nitrate reductase.Nitrogen dioxide detects through colorimetry with the form that the azo-dye product is arranged of the visible light that absorbs about 540nm scope place.The nitric oxide production level that exists in the system is by being converted into nitrogen dioxide with all nitrogen peroxides, measure the nitrogen dioxide total concentration in the unknown sample, then the content of nitrogen dioxide that obtains and nitrogen peroxide with known quantity are converted into the standard curve that produces behind the nitrogen dioxide and compare.

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 to be used for to transplant.

For the inhibition of in-vitro evaluation, measured the inhibiting value relevant with endothelial cells cultured to smooth muscle cell growth.With in pig or the sparse smooth muscle cell growth culture medium that is inoculated in the 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, cultivated 48-72 hour, make the cell growth retardation.To be paved with back endotheliocyte culture and dilute twice by 1: 1, and make conditioned medium, and it is added in the smooth muscle cell culture with the SMC growth medium.Each test includes the positive control that suppresses smooth muscle cell growth, for example heparin.After 3-4 days, the cell number in every duplicate samples is counted with the Coulter enumerator.Add before the conditioned medium and be exposed to conditioned medium and control medium (standard growth culture medium by relatively facing, add or do not add somatomedin) following 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 with the conditioned medium sample and positive control are relevant compares.According to preferred embodiment,, but think that then this implantable flow composition has inhibition if the suppression ratio of conditioned medium reaches suppression ratio about 20% of heparin contrast.

Be the suppression ratio of in-vitro evaluation, measured the level of the Heparan sulfate relevant with endothelial cells cultured to thrombosis.Heparan sulfate has antiproliferative and antithrombotic property.Conventional dimethylated methylene base indigo plant-chondroitin sulfate lyases ABC photometry or the ELISA method that adopts above-detailed to cross calculates per 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 is 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 with platelet rich plasma.At room temperature in the pig blood sample, add sodium citrate, 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 endotheliocyte culture preparation condition culture medium after being paved with, and add in the platelet rich plasma aliquot.In blood plasma, add platelet aggregating agent (agonist) in contrast.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).The blood plasma aliquot that another part do not add platelet agonist or conditioned medium is 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).Measure the platelet aggregation that obtains under all test conditions with the gathering instrument then.Assemble instrument and measure platelet aggregation by monitor optical densities.Work as platelet aggregation, then have more light can pass through sample.Assemble of function " platelet aggregation unit " expression of instrument results reported with platelet aggregation speed.Measure maximum the gathering when adding behind the agonist 6 minutes.By reaching the platelet aggregation of positive control after comparing the baseline platelet aggregation of platelet rich plasma before adding conditioned medium and being exposed to conditioned medium, determine the effect of conditioned medium to platelet aggregation.The result represents with the percent of baseline.The inhibiting value that inhibiting value that will be relevant with the conditioned medium sample and positive control are relevant compares.According to preferred embodiment, about 20% time when the suppression ratio of the positive contrast of suppression ratio of conditioned medium, but can think that then this implantable flow composition has inhibition.

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

According to another alternate embodiment, but implantable flow composition is drawn to the syringe from culture tube, adds the 1-20ml culture medium, or the culture medium of enough volumes is transported together and preserved, add the medicated 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 improved through the filter medicated cap, 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, preferably the endotheliocyte of implanting in the endothelial cell growth culture medium of about 50ml adds up to about 7 * 10 ⁵To about 1 * 10 ⁶Individual.Total cell number that each patient accepts is preferably about 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 about 90%, and preferred density is about 1.4 * 10 ⁴-2.1 * 10 ⁴Individual cell/mg microgranule; When being paved with or closely be paved with, the heparin that conditioned medium comprised that described cell produces is at least about 0.5-1.0mg/10 ⁶Individual cell/sky is preferably at least about 1.0mg/10 ⁶Individual cell/sky.TGF-β in the conditioned medium ₁Be at least about 200-300picog/ml/ days, preferably at least about 300picog/ml/ days; B-FGF in the conditioned medium is lower than about 200picog/ml, preferably is not more than about 400picog/ml.

According to another embodiment, but in flow composition, adding one or more other materials before the 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 the essential condition that keeps the cell integrity when handling flow composition.Be unexpectedly, can not have carrier or other to be commonly used to protect the reagent of cell integrity in the cell preparation as described herein, or be commonly used to improve processing and reduce the non-solid splitted reagent of preparation that maybe can flow that causes by shearing force.

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

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

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

For estimating the effect of other candidate's carrier fluid, adopt high microgranule that the carrier ratio is carried out preliminary test, the minimum of required carrier when producing a desired effect with mensuration.The Expected Results of estimating comprises, for example improves to handle and improve the mobile of compositions and keep the survival rate and the effectiveness of cell.In addition, but also study with test and under the condition that does not influence cell survival rate or function, the implantable compositions of plane is converted into the ability of implantable particle type flow composition (referring to co-pending application PCT/US______, be also referred to as ELV-002PC, its full content is incorporated herein by reference).For example, in the initial test solution, the 50mg microgranule only contains the carrier solution of the 0.1-1% that has an appointment.It is to contain about 10% carrier in the 50mg microgranule that this concentration increases to maximum with incremental mode.

Subsequently in the test of carrying out, carrier fluid is during from microgranule hydration or cell inoculation, in whole incubation, add incrementally, but reach when being paved with and preparing transportation and/or bestowing the patient when the cell in the implantable flow composition with regard to making like this, the concentration of the carrier fluid in the implantable material during from early stage cell attachment the acceptable carrier fluid ratio of hanging down of microgranule has been increased to higher desired proportions.For example, according to exemplary scheme, when initially hydration and each culture medium were subsequently changed, the carrier solution with 0.1% joined in the microparticle material, was 1% carrier solution up to the acquisition ultimate density.We think, though all can have some cells to run off each when introducing carrier fluid, but most cells sneak in the implantable flow composition of vehicle treated, and can under this environment, grow to and be paved with or closely be paved with.

Carrier includes but not limited to that rare glycerol, alginate (preferred about 1% alginate soln), glucosan or dextrose (the preferably glucosan of about 1-10% or dextrose solution), other steamed bun stuffed with sugar draw together for example glucose, sucrose and fructose, starch (preferably about 6% hydroxyethyl starch solution), gelatin (the preferably gelatin solution of about 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 used biocompatible matrix type, be transplanted on the 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, can comprise the carrier fluid of by volume about 0.5% to about 10% according to selected carrier.Normally, selected carrier should be under used dosage and concentration no cytotoxicity, and have sufficient permeability, to allow air and nutrient substance to flow into (with the sustenticular cell growth) and to flow out (with discharge cell waste product) implantable compositions.

The influence of undiluted glycerol carrier pair cell survival rate: the porcine aorta endotheliocyte is inoculated on the 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, be placed on then in the syringe that is connected with 24,26 or No. 27 pins with the undiluted glycerol aliquot of 3ml.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% after discharge, to survive 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, obviously shown this carrier fluid under undiluted situation as single dose to being paved with the adverse effect that cell adds fashionable pair cell survival rate.Do not determine to use this carrier fluid whether to help keeping being paved with of cell monolayer at present as yet.

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

According to an embodiment of method of modifying, in case reaching on the implantable compositions of plane, cell is paved with, then compositions is transferred to syringe.For adapting to this plane form, syringe can be with pin or have big 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 from syringe, to pass through.For example, material can then by big vent needle, then pass through the more pin of aperture earlier by there not being the syringe of pin, reaches the particle diameter and the flowability of expection until material.Repeatedly by and the increment of particle diameter modified the injury amount that can advantageously reduce pair cell during such material is modified, 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 contacting, perhaps be applied to outer surface, when perhaps along the circumferential direction using with arc with the outer surface of tubular structure, compositions can also effectively seal the abutment of two or more tubular structures, perhaps the 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 intracavity skin lesion to hinder.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 the 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 the composition that supports endothelium or the needed characteristic of endothelioid cells group simultaneously, such as but not limited to the fibrin net.Similarly, biocompatibility substrate itself can have the sealant characteristic simultaneously and support the needed characteristic of cell mass.Under the situation of other embodiment, the functional of sealant can be provided by cell at least in part.For example, expection produces with the bonded cell of compositions can modify the material of substrate, thereby makes substrate obtain the sealant characteristic, also shows/keep the cell function that it is essential simultaneously.Some cell can produce such material naturally, and other cell can produce such material by transforming the back.

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

But the freezing preservation of implantable flow composition: but comprise the implantable flow composition that is paved with, closely is paved with or is paved with the back cell mass can freezing preservation with storage and/or be transported to clinical mechanism, and after last thawing, do not reduce its clinical efficacy or integrity.But implantable flow composition is preferably at 15ml cryovial (Nalgene ^, Nalge Nunc Int ' l company, Rochester, the New York) in, freezing preservation in the solution that contains have an appointment 5ml CryoStor CS-10 solution (BioLife Solutions company, Oswego, New York), about 10% DMSO, about 2-8% glucosan and about 50-75%FBS.Cryovial is placed the cold isopropanol water-bath, be transferred in-80 ℃ of refrigerators 4 hours, be transferred to then in the liquid nitrogen (150 to-165 ℃).

About 15min that then but the aliquot of the implantable flow composition of freezing preservation at room temperature slowly thawed then thawed in room-temperature water bath about 15 minutes again.Then material is washed about 3 times with culture medium (wash media) with about 15ml washing.Washing comprises the no phenol red EBM that contains or do not contain 50 μ g/ml gentamycins with culture medium.Initial twice cleaning procedure at room temperature carried out about 5 minutes.Last cleaning procedure is at 37 ℃, 5% CO ₂Under carried out about 30 minutes.

Thaw and cleaning procedure after, the material that makes freezing preservation about 48 hours of dormancy 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 back adjusting of further thawing was carried out other at least 24 hours before can and/or packing in use and being used to preserve or transport.

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 about 50ml, sustenticular cell reaches 14 days under the condition of not changing culture medium.Before the transplanting, decant goes out unnecessary culture medium, and, if having serum in the transportation culture medium, but then 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 dispersion, and therefore lost cell is paved with state, so in last cleaning procedure, said composition should be retained in the cask.In addition, but preferably can keep the improvement of the integrity of implantable flow composition in operation to the improvement of cask, described improvement includes but not limited to filter and/or independent cultivation compartment.

But the implantable flow composition of flushing for several times after, but the flushing liquor that keeps about 1-3ml absorbs this material to delivery apparatus for example in the syringe helping at the top of implantable flow composition.Operate delivery apparatus then, but implantable flow composition is sucked in the delivery apparatus, careful as far as possible during operation, the destruction that is paved with cellular layer is minimized.According to another embodiment, the loading attachment of materials used for example at the cask opening with send with the infundibulate interface between the syringe, reduces material cell breakage in being transferred to the process of delivery apparatus.To delivery apparatus, the about 1ml of discharge section remaining liq to be ready to delivery apparatus, fills 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 is about 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 to the cell by pin, by the pin of internal diameter in these scopes.Surprisingly, cell preparation can not influence or damage under the state of being paved with or the functional condition picked-up and be No. 21 (internal diameter=0.019 inch) to No. 30 (internal diameter=0.006 inch) from scope, flows out in the pin hole of preferred No. 24 (internal diameter=0.012 inch).Equally surprisingly, cell is by after the pin, and is only very good as performance under the condition of carrier fluid with cell growth medium.

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

Table 1

By No. 22 pins

Experimental result	No. 22 pins	Needleless
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	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	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
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	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	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 biological output of cell do not have harmful effect.According to preferred embodiment, cell inoculation is on matrix of microparticles, and propagation is to being paved with.Be paved with back 3 days, but with flow composition by inside diameter ranges be about 0.007 inch to about 0.018 inch sterile needle conduit, collect, recovery is 24 hours in endothelial cell growth culture medium-2 (EGM-2).After convalescent period, made culture medium conditionization 24 hours.Then to estimating by the conditioned medium of the cell behind the pin.Conditioned medium after passing through is estimated its product basic fibroblast growth factor (b-FGF), Heparan sulfate (HS) and transforming growth factor ₁(TGF-β ₁) acceptable level.In addition, carry out the smooth muscle cell test to show the inhibitory action of culture medium to smooth muscle cell proliferation.The experimental result of cell that will be by pin compares (seeing Table 2) with the result of the cell of the implantation microparticle material sample by wire guide not.

Table 2

Pass through wire guide

Experimental result	Wire guide	No wire guide
Experimental result	Wire guide	No 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	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/mL	360	336
bFGF pg/mL	58	61.7	TGF-β ₁ pg/mL	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 can insertion by the device of treatment blood vessel.Endoscope navigation system can be used for delivery apparatus is positioned medicine-feeding part, comprises that intravascular ultrasound (IVUS) for example, color doppler ultrasonography, dual-functional ultrasonic, other routine are ultrasonic, fluoroscopy and/or other endoscope navigation system well known in the art of 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 sacculus angioplasty or support or other device combines, before other blood vessel internal program, simultaneously or carry out afterwards.

In one embodiment, be equipped with on the intraluminal delivery device and cross or penetrating device, this device can pass the lumen of vessels wall, arrives the surface, non-chamber of blood vessel.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 then.

The surface, non-chamber (also being called outside the chamber) that this paper relates to can comprise the 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, the position is any surface except the inner surface in chamber outside non-chamber or the chamber.

Penetrating device that this paper relates to allows for example single delivery sites, perhaps a plurality of delivery sites of arranging with the expection geometric configuration, but under the condition of not disturbing impaired or ill target site, finish sending of flow composition surface, vasotropic non-chamber.A plurality of delivery sites can be by for example 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 or near the position topical this position with this position.But the sedimentary position of flow composition is outside the chamber.As this paper estimated, the local deposits outside the chamber can as mentioned belowly be finished 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, the sending of position to the needs treatment is 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 that intravascular ultrasound (IVUS) for example, color doppler ultrasonography, dual-functional ultrasonic, other routine are ultrasonic, fluoroscopy and/or other endoscope navigation system well known in the art of 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.Behind the intravasation week crack, but the doctor is deposited on the position chamber outside with flow composition, and the position is on the position that needs are treated this chamber outside, 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, outside this chamber the position on the position of needs treatments, or adjacent, or near the position of needs treatment with the position of needs treatment.Finish at the position that orientation of sending in this case, and guide need be treated by direct observation.Equally in this case, can use authentication step mentioned above to help to send simultaneously.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 to be had 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 of being treated, or impaired or disease sites is adjacent with this, or at this near the impaired or disease sites.Compositions can be in all places deposition relevant with impaired or ill target site, for example, on impaired or ill target site, with impaired or ill target site position adjacent on, 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 is from the position of the about 2mm of impaired or ill target site to about 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 sedimentary compositions can show expected effect in this position.

According to an embodiment, but before implantable flow composition administration, outside the chamber, make plane administered area arbitrarily in the target site.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 use, vertical with blood flow direction; Or be block at medicine-feeding part.But sending of aforementioned dissection step and flow composition also can take place simultaneously or take place sequentially.For example, if under pressure, send,, implantable flow composition self dissects but just can being used for finishing fluid.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 finishing blunt dissection, when delivery apparatus when the plane administered area of new generation is recalled, but promptly finished 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 an 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 an 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's body.

The endoscope navigation system at other positioning chamber external administration position includes but not limited to that color doppler ultrasonography, dual-functional ultrasonic, other routine are ultrasonic, fluoroscopy and/or other endoscope navigation system well known in the art of 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 the administration.According to an embodiment, but video picture has been delivered to chamber external series gap rather than intracavity gap to guarantee implantable flow composition after carrying out administration.

Dosage: in a preferred embodiment of the invention, have an appointment 1 * 10 in the volume of the implantable microgranule of about 50-60mg but each flow composition that can send administration contains 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, carries out the single or multiple administration to a plurality of therapentic parts, and/or provides administration during single treatment incident even in the long-term treatment process.

But the administration of each flow composition includes voidage, and voidage is to stay the part of the appointment administration volume in the delivery apparatus after the administration.But the scope of voidage be loaded into delivery apparatus the flow composition volume about 1% to about 50%.Consider voidage, the amount that is loaded into the cell of delivery apparatus and microgranule is greater than the actual dosage that is intended to bestow the patient.Certainly, but the 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 when single therapentic part is implemented multiple dose administration, the dosage of expection can be loaded into single delivery apparatus, and bestows in single-dose.Yet,, repeatedly independently finish heavy dose of administration in the administration when need when a plurality of therapentic parts of single patient are implemented multiple dose administration, preferably using the fixed aliquot of sending product of many umbers amount.The advantage of fixedly administration aliquot comprises that it is inaccurate that minimizing is divided into a plurality of low dose of dosage of introducing by a heavy dose; The dosage that minimizing is introduced by the limitation of the inherent 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 littler aliquot require compositions is carried out unnecessary processing, comprise and destroy adjacent granule, destroy other damages that are paved with cell monolayer and cause pair cell.

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

Reflux: tried body with the single pig of accepting Gelfoam microgranule intravascular administration and carry out preclinical study.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 that is tried 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 take place to lumen of vessels.In addition, carry out subsequently to not showing that through the painted Histological evaluation of being cut into slices of VerhoeffShi elastin laminin staining suspension overflows the intravasation chamber or enters the evidence of surrounding tissue from adventitia by treated tissue.

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

Embodiment 1: animal blood vessels gets involved research

Present embodiment provides animal and has been tried 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 body.Adopt SOP, by pass through ball capsule angioplasty and insert support and cause the exocoel surface damage at the femoral artery place.Then but implantable flow composition of the present invention is deposited on position adjacent blood vessel week with angioplasty and stent in the treatment blood vessel in the crack; The details of an exemplary program is set forth hereinafter.As previously mentioned, but implantable flow composition insert with preparation can be miscellaneous.

This research specifically comprises 26 pig experimenters that accept percutaneous sacculus angioplasty and Stent.Carry out traditional percutaneous sacculus angioplasty and Stent program according to the standard operation technology.Finish angioplasty and stenting, and setting up by by behind the blood flow of treatment blood vessel, but with implantable flow composition the position that balloon expandable and support insert and on every side of being applied to as mentioned below.

Operative procedure: accept the experimenter of percutaneous sacculus angioplasty and Stent for each, all under dorsal position, accept intubate and link to each other with the heart monitor controller.Introduce 7French sheath pipe through the incision approach to the right carotid inlet, 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 finished and record by cineradiography.Under 8-10 atmospheric pressure, expand 30 seconds by sacculus, (whenever 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.

At angioplasty and after inserting support, but comprise endotheliocyte and matrix of microparticles, only comprise blood vessel that matrix of microparticles or flow composition not to be covered whatever be delivered to left side and right common femoral artery through pumping needle conduit (needle injection catheter) orientation on every side.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 position in the about 1-20mm of mount proximal end for example in the mount proximal end position, and the rack far end position is for example in the about 1-20mm of rack far end, and along the position administration of support total length.Each injection position is all accepted about 0.1-1.0ml, comprises about 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 kind.These 6 experimenters are used for comparing with nursing standard.Total cell carrying capacity is about 1 * 10 according to body weight ⁴-8 * 10 ⁴Individual cell/kg.

But finish the angiopoiesis program, support is inserted and implantable flow composition after the injection in blood vessels adjacent week crack, C type arm cryptoscope is placed experimenter's cervical region top, make by the treatment angiography.Under successive fluoroscopy, injection 10-15cc iodine contrast medium (Renograffin, full concentration).Record and store movies angiography, the angiogram preceding with execution compares.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 give heparin 35U/kg/h and continue to operation to finish through continuous infusion.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 the operation and measure,, carried out once again, and after this carried out once weekly in postoperative 3-7 days to determine to be treated the patency rate of blood vessel.Monitor closely is treated the blood flow of blood vessel.Must detect flow by blood vessel, after the experimenter in being placed in research undergos surgery the 7th day, and comprise the 7th day.If do not detect flow before the 7th day or the 7th day the time by blood vessel, then 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) implemented euthanasia in back 1 month in operation.

Animal subjects is anaesthetized with pentobarbital sodium (65mg/kg, intravenous injection).Expose by the treatment blood vessel, carried out digital camera treating blood vessel and surrounding tissue and blood vessel structure.Then C type arm cryptoscope is placed animal's neck, make by the treatment angiography.Under successive fluoroscopy, injection 10-15cc iodine contrast medium (Renograffin, full concentration).Treated 0 ° and 90 ° of record cineangiographies of blood vessel.By blind reading postmortem angiogram, carry out paired comparison and determine vascular patency and narrowness with angiogram after inserting.Angiogram is according to being divided into the 0-5 level in the observed narrowness of 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 after but the blood vessel of implantable flow composition treatment of the present invention is being checked angiogram and contrast the experimenter and compare the narrowness that reduces.

The histology: the animal subjects to half (is received treatment for 5; 5 contrasts; 3 experimenters do not accept implantation) implemented euthanasia in back 3 days in operation.Remaining animal subjects (is received treatment for 5; 5 contrasts; 3 experimenters do not accept implantation) implemented euthanasia in back 1 month in operation.

The postmortem that all experimenters all limit limits postmortem and is defined as macroscopy all experimenter's medicine-feeding part and surrounding tissues, comprises draining lymph node.Experimenter for euthanasia is implemented in all operations after 1 month collects and preserves the tissue that its major organs comprises brain, lung, kidney, liver, the heart and spleen.Have only when occurring difference in the macroscopy of the macroscopy of body outer surface or medicine-feeding part and surrounding tissue and find, just organ is analyzed.

All are treated blood vessel and surrounding tissue excision, in stuck-at-0% formalin (or equivalent), and be embedded in the glycolmethacrylate (or equivalent).Be cut into about 3 μ m slabs with the stainless steel knife of C type (or equivalents), the section of preparation is taken from by three fragments of treatment blood vessel: 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 the smooth muscle cell and the endotheliocyte of lumen of vessels around the blood vessel is marked.Other tissue slice available from 1 month experimenter dyes with the VerhoeffShi elastin laminin and checks, vessel size, blood vessel injury degree, neointimal hyperplasia and restenosis are marked.Other section also can include but not limited to PECAM-1 and α-SMC actin with specificity endotheliocyte and the dyeing of smooth muscle cell labelling.Check the residual blood tube chamber simultaneously, with the geometric change of reflection blood vessel after impaired and reparation.Adopt computerized digital planimetry to carry out the form quantitative analysis with videomicroscopy and customized software the section of Verhoeff Albert'stain Albert again.

The blood vessel of determining acute (3-5 days experimenters) and chronic (1 month experimenter) reaches the lumen of vessels inflammation on every side.The acutely inflamed granulocyte that is masked as mainly is a 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 to discern leukocyte, and anti-CD3 is used to discern the T cell, and CD79 is used to discern the B cell, and MAC387 is used to discern monocyte/macrophage.

Check the slide after dyeing, the existence of smooth muscle cell and endotheliocyte is marked.To by the treatment blood vessel all partly, comprise that the middle film outside (adventitia position) of the middle film inside of inner membrance/false inner membrance, hematochezia tube chamber, nearly adventitia and adventitia estimate and mark.Be that unit measures each and organizes for example size of inner membrance, middle film and adventitia of compartment with the micron.Each section is estimated with regard to the 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 the evaluation of tissue section, new vessels, calcification, hemorrhage, congested, fibrin, graft fibrosis and graft permeate.In addition tissue slice is carried out the degeneration evaluation of indexes, 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 cell proliferation under tissue slice evaluation endothelial cell proliferation, the inner membrance is included 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 is all given branch by the grade of 0-4, and (0=does not have obvious change; 1=is slight; 2=is slight; The 3=moderate; And 4=severe).

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

If when checking blood vessel roughly, arrive local damage, blood vessel wall attenuation or diastole in the visual observation at above-mentioned position, then can also under pathologist's judgement, obtain other section.Check with the slide of blind (reading) mode after and mark by the veterinary pathology person of specialty authentication all dyeing.

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 indicated 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 lumen of vessels and/or the minimizing of periangiitis disease.

Another index of successfully treating 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 treatment was condemned to death at same day and facing on the 30th day with the angiography monitoring by the lumen of vessels diameter and the narrow percentage rate of treatment blood vessel.The lumen of vessels postoperative is narrowed down and rate of blood flow connects with standard doppler ultrasound scheme.Expection the present invention can prevent or postpone to narrow down, thereby stop rate of blood flow to be 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 by rack far end and the occluding thrombus disease of mount proximal end vasculature part or the narrow generation and/or the degree (so-called edge effect) of treatment blood vessel.Edge effect or candy wrapper effect are meant that support inserts the back in the zone of support junction (articulations) and the narrow development in edge.Edge effect is narrow with early stage new intima hyperblastosis and late period to be feature, can cause occluding thrombus disease.Before treatment was condemned to death at same day and facing on the 30th day, with angiography to being monitored by the edge effect of treatment blood vessel.The present invention can be as described herein such prevent or relevant thrombosis and the blood vessel of edge effect that delay and support are inserted blood vessel narrows down.The existence of edge effect or disappearance also can be cut into slices by obtaining utmost point near-end and utmost point far-end, i.e. the section at the about 2-3mm in the upstream at the two-way edge of support or downstream place, and it is carried out histological examination determine.Be the existence of evaluation edge effect, damage, inflammation, new intima formation and the thrombosis of utmost point near-end and the section of utmost point far-end are marked.

Quilt treatment subject group is compared with matched group has at least a kind of aforementioned functional index to demonstrate the difference of increment type at least.

Embodiment 2: people's blood vessel is got involved research

But 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, finish percutaneous sacculus angioplasty and stenting, to alleviate clinical disease by doctor's control.Then but implantable flow composition is deposited on the perivascular canal at angioplasty and stenting position, perhaps with angioplasty and all cracks of stenting position adjacent blood vessel, perhaps near the perivascular canal angioplasty and stenting position; The details of an exemplary program is set forth hereinafter.As previously mentioned, but inserting of implantable flow composition with preparation can change in the usual way by skilled practitioner.

Especially, this research is included in the people experimenter that periphery limbs (peripheral limb) are accepted percutaneous sacculus angioplasty and stenting.Carry out traditional percutaneous sacculus angioplasty and stenting operation according to the standard operation technology.Finish angioplasty and stenting, and setting up by by behind the blood flow of treatment blood vessel, but with the implantable flow composition of the present invention position of balloon expandable and on every side of being applied to as indicated above.Total cell carrying capacity is about 1.0 * 10 based on body weight ⁴Individual cell/kg is to about 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, 2 weeks, 1 month, 3 months and 6 months are carried out blood flow measurement after surgery then.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% (measuring with doppler ultrasound).Permission determines that to the intravascular radiography experimenter that narrow sexually transmitted disease (STD) becomes greater than 50% carries out for example angioplasty of the clinical intervention of therapeutic.

Compared angiography baseline day and 3rd month to treating blood vessel and surrounding tissue and blood vessel structure.Calculate each regional lumen of vessels diameter, measure the peak systolic blood flow rate.

People's blood vessel is got involved the result of research

As indicated 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.In baseline day (about 5 days of treatment back), the lumen of vessels diameter of with angiography quilt being treated blood vessel at least 3 months is after surgery monitored then earlier.The lumen of vessels postoperative is narrowed down and rate of blood flow connects with standard doppler ultrasound scheme.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 to be lower than the speed that is suitable for peripheral circulation described herein.Further, but can produce the acceptable clinically circulation rate of blood flow of permission with implantable flow composition treatment of the present invention, or near the rate of blood flow of normal speed.It is comparable flowing into or flowing out by the blood flow of treatment part blood vessel.The comparable meaning and clinical application broadly similar.For example, rate of blood flow is about 150-500mL/min, preferably about 300-500mL/min, more preferably from about 350-400mL/min.

Embodiment 3: the adhesion research again of animal pelvis

But 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, to reduce the experimental program that pelvis and adhesion on every side thereof take place.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.But as indicated above with implantable flow composition of the present invention be 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 the 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

Present embodiment provides in animal subjects test and has used the 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.From the active electrode conduit, stretch out wire guide, carry out the RF electric coagulation.But as indicated above with implantable flow composition of the present invention be applied to an oviductus lateralis and around.But the opposite side fallopian tube is not accepted implantable flow composition, in contrast.Constantly monitoring fallopian tube 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 are in the implication of the equivalents of this claim and the variation in the scope, all therefore will comprise in the present invention.

Claims

1. treat the flowable compositions of the impaired or disease sites of tubular anatomical structures, wherein said flowable compositions comprises biocompatible matrix and cell, and the amount of described flowable compositions is the amount of effectively treating impaired or disease sites.

2. the flowable compositions of claim 1, wherein said cell are endotheliocyte or cell with endotheliocyte sample phenotype.

3. the flowable compositions of claim 1, wherein said cell is two or more coculture that is selected from the cell of endotheliocyte, epithelial cell, smooth muscle cell, fibroblast, stem cell, endothelial progenitor cells and myocardial cell.

4. the flowable compositions of claim 1, flowable compositions wherein are to keep shape.

5. the flowable compositions of claim 1, biocompatible matrix wherein comprises microgranule or microcarrier.

6. the flowable compositions of claim 5, microgranule wherein or microcarrier further comprise gelatin, collagen protein, fibronectin, fibrin, laminin or attaching peptide.

7. the flowable compositions of claim 6, attaching peptide wherein comprises the peptide of sequence arginine-Gly-Asp (RGD).

8. the flowable compositions of claim 5, the microgranule wherein or the diameter of microcarrier are about 20 microns to about 500 microns.

9. the flowable compositions of claim 5, the microgranule wherein or the diameter of microcarrier are about 200 microns.

10. the flowable compositions of claim 1, it further comprises cell growth medium.

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

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

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

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

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

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

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

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

19. the method for the impaired or disease sites of treatment tubular anatomical structures, this method may further comprise the steps:

Make the chamber outer surface of described tubular anatomical structures and flowable compositions impaired or disease sites in described tubular anatomical structures, or it is adjacent with this position, or near this position, contact, wherein said compositions comprises biocompatible matrix and cell, and the amount of wherein said flowable compositions is the amount of effectively treating impaired or disease sites.

20. the method for claim 19, deposition wherein is by passing or see through the inwall of described tubular anatomical structures, then at impaired or disease sites, or adjacent with this position, or near this position, the flowable compositions is deposited on the outer surface of described tubular anatomical structures and finishes.

21. the method for claim 20, it further comprises identifies the step of flowable compositions at the position of the outside deposition of described tubular anatomical structures.

Pass or see through before the step 22. the method for claim 21, authentication step wherein occur in, perhaps with pass or see through step and take place simultaneously.

23. the method for claim 21, authentication step is wherein finished by imaging.

24. the method for claim 21, authentication step is wherein finished by palpation.

25. the method for claim 19, wherein said contact procedure is passed through percutaneous dosing intravasation week crack, then at impaired or disease sites, or adjacent with this position, or near this position, the flowable compositions is deposited on the outer surface of described tubular anatomical structures and finishes.

26. the method for claim 25, it further comprises identifies that the flowable compositions is deposited on the step at position of the outer surface of described tubular anatomical structures.

27. the method for claim 26, authentication step wherein occur in and enter before the step, perhaps with enter step and take place simultaneously.

28. the method for claim 26, authentication step is wherein finished by imaging.

29. the method for claim 26, authentication step is wherein finished by palpation.

30. the method for claim 19, the surface, outer surface right and wrong chamber of wherein said tubular anatomical structures.

31. the method for claim 19, the outer surface of wherein said tubular anatomical structures has occupied perivascular canal.

32. the method for claim 19, wherein said tubular anatomical structures is a blood vessel.

33. the method for claim 32, wherein said blood vessel comprises support.

34. the method for claim 33, wherein said impaired or disease sites is near support.

35. the method for claim 19, wherein said effective dose reduces the propagation of the smooth muscle cell of impaired or disease sites.

36. the method for claim 19, wherein said effective dose alleviates the occluding thrombus of impaired or disease sites.

37. the method for claim 19, wherein said effective dose alleviates the neointimal hyperplasia of impaired or disease sites.

38. the method for claim 19, wherein said effective dose alleviate the narrow or restenosis of impaired or disease sites.

39. the method for claim 19, wherein said effective dose alleviates the acute inflammation of impaired or disease sites.

40. the method for claim 19, wherein said effective dose alleviates the chronic inflammatory disease of impaired or disease sites.

41. the method for claim 19, wherein said effective dose alleviate the vasodilation or the vasospasm of impaired or disease sites.

42. the method for claim 19, wherein said tubular anatomical structures is a fallopian tube.

43. the flowable compositions of claim 1, wherein said cell is selected from: the cell mass that is paved with; Near the cell mass that is paved with; Cell mass after being paved with; And any cell mass with aforementioned phenotype.