CN101534747A

CN101534747A - De novo formation and regeneration of vascularized tissue from tissue progenitor cells and vascular progenitor cells

Info

Publication number: CN101534747A
Application number: CNA200780026277XA
Authority: CN
Inventors: J·J·毛
Original assignee: Columbia University in the City of New York
Current assignee: Columbia University in the City of New York
Priority date: 2006-07-10
Filing date: 2007-07-10
Publication date: 2009-09-16

Abstract

It has been discovered that vascularized tissue or organs can be engineered by combined actions of tissue progenitor cells and vascular progenitor cells. Provided herein are compositions and methods directed to engineered vascularized tissue or organs formed by introducing tissue progenitor cells and vascular progenitor into or onto a biocompatible scaffold of matrix material. Also provided are methods of treating tissue defects via grafting of such compositions into subjects in need thereof.

Description

From organizing CFU-GM and blood vessel CFU-GM from the beginning to form and the regeneration vessel tissue

The cross reference of related application

The application requires the priority of the U.S. Provisional Application No.60/824597 that the U.S. Provisional Application No.60/819833 that submitted on July 10th, 2006 and JIUYUE in 2006 submitted on the 5th, and these two parts application integral body are incorporated into herein for your guidance.

Subsidize the statement of research and development about federal government

A part of the present invention is to utilize the government according to national biomedical imaging and bio-engineering research institute (National Institute of Biomedical Imaging and Bioengineering) and national dentistry and cranium face institute (National Institute of Dental and Craniofacial Research) research foundation No.R01DE15291 and R01EB02332 to support to carry out.Government has certain right to the present invention.

Incorporate the material of submitting on the compact disk into by reference

No.

Invention field

The present invention relates on the whole from organizing CFU-GM and blood vessel CFU-GM from the beginning to form and the regeneration vessel tissue.

Background of invention

For be used for wound reconstruction, chronic disease, tumor removes and the clinical demand of the tissue transplantation of birth defect is a large amount of.Present surgical procedure depends on autotransplantation, allograft, xenotransplantation or synthetic material.Surgery has extensively been recognized the defective relevant with present clinical procedure with scientific research group.

Need limit the development of transplantable clinically three-dimensional engineering (engineered) tissue or organ through dabbling vascular bed with the fact of supplying with nutrient and removing refuse, metabolic intermediate and secretions greater than the tissue assembling of 100-200 μ m.Consider that vascular development is complicated incident, it relates to various cell types and many different growth factors, and sophisticated functional blood vessel network has been difficult to build.In the process of fetal development, endotheliocyte forms pipe and in conjunction with forming primary capillary plexus, this process is known as angiogenesis.Neovascularity splits in half by existing blood vessel or forms by sprouting from existing blood vessel.In being known as the process of mature blood vesselization, this elementary network is retrofited comprises capillary tube, tremulous pulse and vein with pruning to form different microcirculation units.

The angiogenesis of suboptimum remains the significant obstacle of organizational project, particularly for the significant dimensions tissue defects.The approach of angiogenesis engineering relies on the release of angiogenesis growth factor or the manufacturing of blood vessel analog before.Yet, exist cost to delivery of growth factor, genotoxic potential, suboptimum to coincide and endothelial migration is concerned about slowly for big tissue grafts always.

The subgroup that can from single bone marrow sample, separate two kinds of stem cell: mescenchymal stem cell (MSC) and hematopoietic stem cell (HSC).MSC can be divided into most connective tissue pedigree cell.HSC is divided into endotheliocyte, is necessary together with originally hemocyte for the formation of blood vessel tissue.

Therefore, have compositions to engineering blood vessel tissue construct (contruct), with and the demand of production method.

Summary of the invention

Herein disclosed is new method, it is at blood vessel tissue's engineering of the compound action by organizing CFU-GM and blood vessel CFU-GM.The blood vessel tissue's assembly (module) that uses disclosed compositions and method to produce can be used for various clinical and use.

In certain aspects, the present invention relates to blood vessel tissue's assembly.In various configurations, organization component comprises biocompatible matrix, organizes CFU-GM and blood vessel CFU-GM.CFU-GM can be introduced into (for example by while or injection successively, endoscopy or infusion) in the biocompatible scaffold of host material or on it.

Another aspect of the present invention provides a kind of method that forms blood vessel tissue's assembly.These methods comprise provides biocompatible matrix, and organizes CFU-GM and blood vessel CFU-GM to this substrate introducing.Use method as known in the art for example by injection, endoscopy or infusion, CFU-GM can be delivered in the biocompatible matrix or on it.In various configurations, sending can be simultaneously or successively.These methods also further comprise hatches the substrate that contains in a organized way with the blood vessel CFU-GM.In some configuration, in the process of hatching, tissue morphology can take place form and/or cell differentiation.This hatching can be at least partially in external, basically external, at least partially in the body or basically in vivo.In some configuration, can form assembly to small part stripped (ex vivo), and at some in other the configuration, biocompatible matrix, to organize at least a receptor for expectation in CFU-GM and the blood vessel CFU-GM for example to need treatment to carry out for tissue repair or the metathetical people be allogenic.

In many aspects, organizing CFU-GM can be mescenchymal stem cell (MSC), MSC derived cell, osteoblast, chondrocyte, myocyte, adipose cell, neuronal cell, myocardial cell, neurogliocyte, Schwann cell (Schwann cell), epithelial cell, dermal fibroblast, a matter fibroblast, gums fibroblast, periodontal fibroblast, sutura cranii fibroblast, tendon cell (tenocyte), ligament fibroblast, urethra cell, hepatocyte, periosteum cell, β-islet cells or its combination.In some configuration, preferably organizing CFU-GM can be MSC, MSC derived cell or its combination.

In many aspects, the blood vessel CFU-GM can be hematopoietic stem cell (HSC), the HSC endotheliocyte of deriving, vascular endothelial cell, lymphatic endothelial cells, the endotheliocyte pedigree, the former foster endotheliocyte of being commissioned to train, endotheliocyte derived from stem cell, the bone marrow derived stem cell, notochord blood derived cell (cord blood derived cell), Human umbilical vein endothelial cells (HUVEC), the lymph gland endotheliocyte, endothelial progenitor cells, be divided into the stem cell of endotheliocyte, blood vessel CFU-GM from embryonic stem cell, from the endotheliocyte of fatty tissue or periodontal tissue or dental pulp, preferred HSC or the deutero-endotheliocyte of HSC.

In many aspects, substrate can comprise material for example fibrin, Fibrinogen, collagen, poe, polyvinyl alcohol, polyamide, Merlon, agarose, alginate, Polyethylene Glycol, polylactic acid, polyglycolic acid, pla-pcl, polyvinylpyrrolidone, ocean attachment proteins (marine adhesive protein), cyanoacrylate, macromolecule hydrogel, analog or its combination.In some preferably disposed, host material can be a macromolecule hydrogel.

In many aspects, substrate can comprise at least one major path and/or microchannel.In some embodiments, a plurality of major paths can have about at least 0.1mm to the average diameter that is up to about 50mm.For example, major path can have following average diameter: about 0.2mm, about 0.3mm, about 0.4mm, about 0.5mm, about 0.6mm, about 0.7mm, about 0.8mm, about 0.9mm, about 1.0mm, about 1.1mm, about 1.2mm, about 1.3mm, about 1.4mm, about 1.5mm, about 1.6mm, about 1.7mm, about 1.8mm, about 1.9mm, about 2.0mm, about 2.5mm, about 3.0mm, about 3.5mm, about 4.0mm, about 4.5mm, about 5.0mm, about 5.5mm, about 6.0mm, about 6.5mm, about 7.0mm, about 7.5mm, about 8.0mm, about 8.5mm, about 9.0mm, about 9.5mm, about 10mm, about 15mm, about 20mm, about 25mm, about 30mm, about 35mm, approximately 40mm or approximately 45mm.

In many aspects, substrate can comprise at least a somatomedin, preferred angiogenesis growth factor, more preferably bFGF, VEGF, PDGF, IGF, TGFb or its combination.

In many aspects, organization component of the present invention can contain CFU-GM and/or blood vessel CFU-GM in a organized way, and its density is about 0.5 * 1,000,000 CFU-GM (M) ml altogether ^-1～about 100Mml ^-1For example, in various configurations, organization component can contain the CFU-GM of following density: about 1Mml ^-1, 5Mml ^-1, 10Mml ^-1, 15Mml ^-1, 20Mml ^-1, 25Mml ^-1, 30Mml ^-1, 35Mml ^-1, 40Mml ^-1, 45Mml ^-1, 50Mml ^-1, 55Mml ^-1, 60Mml ^-1, 65Mml ^-1, 70Mml ^-1, 75Mml ^-1, 80Mml ^-1, 85Mml ^-1, 90Mml ^-1, 95Mml ^-1Or 100Mml ^-1In some configuration, it is about 0.0001 * 1,000,000 cell (M) ml that organization component can contain density ^-1～about 1000Mml ^-1CFU-GM.In some configuration, it is about 1Mml that organization component can contain density ^-1To being up to about 100Mml ^-1CFU-GM.In some configuration, organization component can contain density and be about at least 5Mml ^-1To being up to about 95Mml ^-1CFU-GM.In some configuration, organization component can contain density and be about at least 10Mml ^-1To being up to about 90Mml ^-1CFU-GM.In some configuration, organization component can contain density and be about at least 15Mml ^-1To being up to about 85Mml ^-1CFU-GM.In some configuration, organization component can contain density and be about at least 20Mml ^-1To being up to about 80Mml ^-1CFU-GM.In some configuration, organization component can contain density and be about at least 25Mml ^-1To being up to about 75Mml ^-1CFU-GM.In some configuration, organization component can contain density and be about at least 30Mml ^-1To being up to about 70Mml ^-1CFU-GM.In some configuration, organization component can contain density and be about at least 35Mml ^-1To being up to about 65Mml ^-1CFU-GM.In some configuration, organization component can contain density and be about at least 40Mml ^-1To being up to about 60Mml ^-1CFU-GM.In some configuration, organization component can contain density and be about at least 45Mml ^-1To being up to about 55Mml ^-1CFU-GM.In some configuration, organization component can contain density and be about at least 45Mml ^-1To being up to about 50Mml ^-1CFU-GM.In some configuration, organization component can contain density and be about at least 50Mml ^-1To being up to about 55Mml ^-1CFU-GM.

In many aspects, blood vessel CFU-GM and the ratio of organizing CFU-GM can for about 100:1 to being up to about 1:100.For example the blood vessel CFU-GM can be about 20:1,19:1,18:1,17:1,16:1,15:1,14:1,13:1,12:1,11:1,10:1,9:1,8:1,7:1,6:1,5:1,4:1,3:1,2:1,1:1,1:2,1:3,1:4,1:5,1:6,1:7,1:8,1:9,1:10,1:11,1:12,1:13,1:14,1:15,1:16,1:17,1:18,1:19 or 1:20 with the ratio of organizing CFU-GM.In some configuration, blood vessel CFU-GM and the ratio of organizing CFU-GM can for about 20:1 to being up to about 1:20.In some configuration, the blood vessel CFU-GM can be 19:1～about 1:19 with the ratio of organizing CFU-GM.In some configuration, the blood vessel CFU-GM can be about 18:1～about 1:18 with the ratio of organizing CFU-GM.In some configuration, the blood vessel CFU-GM can be for approximately with the ratio of organizing CFU-GM.In some configuration, the blood vessel CFU-GM can be about 17:1～about 1:17 with the ratio of organizing CFU-GM.In some configuration, blood vessel CFU-GM and the ratio of organizing CFU-GM can be for approximately in some configurations, and blood vessel CFU-GM and the ratio of organizing CFU-GM can be approximately the 1:16 of 16:1～approximately.In some configuration, the blood vessel CFU-GM can be for approximately with the ratio of organizing CFU-GM.In some configuration, the blood vessel CFU-GM can be about 15:1～about 1:15 with the ratio of organizing CFU-GM.In some configuration, the blood vessel CFU-GM can be about 14:1～about 1:14 with the ratio of organizing CFU-GM.In some configuration, the blood vessel CFU-GM can be about 13:1～about 1:13 with the ratio of organizing CFU-GM.In some configuration, the blood vessel CFU-GM can be about 12:1～about 1:12 with the ratio of organizing CFU-GM.In some configuration, the blood vessel CFU-GM can be about 11:1～about 1:11 with the ratio of organizing CFU-GM.In some configuration, the blood vessel CFU-GM can be about 10:1～about 1:10 with the ratio of organizing CFU-GM.In some configuration, the blood vessel CFU-GM can be about 9:1～about 1:9 with the ratio of organizing CFU-GM.In some configuration, the blood vessel CFU-GM can be for approximately with the ratio of organizing CFU-GM.In some configuration, the blood vessel CFU-GM can be about 8:1～about 1:8 with the ratio of organizing CFU-GM.In some configuration, the blood vessel CFU-GM can be about 7:1～about 1:7 with the ratio of organizing CFU-GM.In some configuration, the blood vessel CFU-GM can be about 6:1～about 1:6 with the ratio of organizing CFU-GM.In some configuration, the blood vessel CFU-GM can be about 5:1～about 1:5 with the ratio of organizing CFU-GM.In some configuration, the blood vessel CFU-GM can be about 4:1～about 1:4 with the ratio of organizing CFU-GM.In some configuration, blood vessel CFU-GM and the ratio of organizing CFU-GM can be for approximately in some configurations, and blood vessel CFU-GM and the ratio of organizing CFU-GM can be approximately the 1:3 of 3:1～approximately.In some configuration, the blood vessel CFU-GM can be for approximately with the ratio of organizing CFU-GM.In some configuration, the blood vessel CFU-GM can be about 2:1～about 1:2 with the ratio of organizing CFU-GM.

Another aspect of the present invention provides the method for treated tissue or organ defective.In various configurations, these methods comprise to be transplanted to organization component of the present invention in the individuality of these needs.

The further aspect of the present invention provides the method for the candidate molecules of identifying the regulation and control tissue blood vesselization.These class methods comprise formation organization component of the present invention; With substrate, organize CFU-GM, blood vessel CFU-GM, its combination or organization component to contact with candidate molecules; Measure the vascularization of engineered tissue compositions; And measure with respect to the contrast that contact with candidate molecules, candidate molecules is regulated and control the formation of engineered tissue compositions (engineered tissue composition) medium vessels.In some configuration, can be before with the combination of substrate and CFU-GM, with cell inoculation is on substrate after but before vascular morphology formation is taking place, perhaps after beginning vascularization, with candidate molecules with substrate, organize CFU-GM or blood vessel CFU-GM to contact.Just as used herein, the regulation and control tissue blood vesselization can comprise compared with the control, improves vascularization or reduces vascularization.

Other purpose and feature are clear with a part, and a part is pointed out.

Description of drawings

It only is for illustrative purposes that those skilled in the art can understand following described accompanying drawing.The scope that these accompanying drawings do not limit the present invention in any way.

Fig. 1 is a series of tissue slice figure, and it has shown that human mesenchymal stem cell (hMSC) is divided into osteoblast.Figure 1A representative is from the bone marrow sample from the preparation of one of a plurality of people's donors, and it has shown a large amount of cells.On behalf of hMSC, Figure 1B become the spindle-type cell from attached cell group amplification cultivation.The MSC that Fig. 1 C representative is handled in the Osteoblast Differentiation culture medium, it shows the positive staining to alkali phosphatase.On behalf of MSC, Fig. 1 D be derivatized to osteocyte, and shown according to Feng Kusa (von Kossa) dyeing, it has produced the mineralising tuberosity.Ratio scale (scale bar): 100 μ m.Further details about this method is provided in embodiment 1.

Fig. 2 is a series of figure, and it shows from epithelial cell that is derived from human mesenchymal stem cell (hMSC) and osteoblastic engineering bone construct.Fig. 2 A representative is inoculated into (TCP: pale red) in the hole of tricalcium phosphate derived from the osteoblast of hMSC.Under 4 ℃, Human umbilical vein endothelial cells (HUVEC) increases and is inoculated in the basement membrane hydrogel, at the Matrigel of aqueous phase, and is infused in the hole of TCP only, then 37 ℃ of following Matrigel gelations.Fig. 2 B is presented at the area that body is implanted into the class osseous tissue (B) in the TCP zone in the sample that obtains afterwards in the immunodeficient mouse back.H﹠amp is used in Fig. 2 C representative; The painted section of E, it shows the chamber that forms by round cell centered on.If HUVEC is seeded among the aqueous Matrigel equably, then in the formation of this construct inner chamber and primitive vessel sample (PV) structure, there is the significantly reconstruction of the HUVEC that inoculates.The more Feng Kusa dyeing of high-amplification-factor is used in Fig. 2 D representative, and it is presented at TCP mineralized tissue island on every side.Ratio scale: 100 μ m.Further details about this method is provided in embodiment 1.

Fig. 3 is a series of figure and block diagram, and it shows that hematopoietic stem cell is divided into endotheliocyte towards engineering vascularization bone.Fig. 3 A represent isolating bone marrow, CD34+, non-adherent cell be laid down on the cell culture polystyrene plate that applies fibronectin.Although these cells are to separate from identical bone marrow with MSC shown in Figure 1, the form of HSC is circular, and the MSC among Figure 1B is fusiform.Two weeks back formation colony is being cultivated in Fig. 3 B representative.Fig. 3 C demonstration is seeded in the Matrigel formed tubular structure between unconjugated cell by the HSC that will form colony.Fig. 3 D shows to locate in the cell as Ac-LDL fluorescence to be proved, to the positive mark of acetylation low density lipoprotein, LDL (Ac-LDL).Fig. 3 E shows that the label-von willebrand's factor (vonWillebrand Factor) of deriving HSC endothelium class cell also expressing a kind of natural endotheliocyte (vWF): Fig. 3 F proof HSC endothelium class cell of deriving produces the vWF (left post) of significantly Duoing than control cells (fibroblast) (right post).Further details about this method is provided in embodiment 2.

Fig. 4 is a series of sketches, and it shows the structure of PEG hydrogel.Fig. 4 A representative only has the PEG hydrogel, does not have bFGF or major path.Fig. 4 B represents the PEG hydrogel, has 3 major paths (diameter 1mm) that form after photopolymerization, but does not have bFGF.Fig. 4 C represents the PEG hydrogel, and it is the 10ug/ml bFGF in the filling solution after photopolymerization, but does not have major path.Fig. 4 D represents the PEG hydrogel, and it has 10ug/ml bFGF and adds major path.From people such as Stosich (2006).Further details about this method is provided in embodiment 3.

Fig. 5 is a series of photos, and it describes to gather in the crops the sample that body is implanted into.Fig. 5 A shows the PEG hydrogel gathered in the crops, it does not have cell, bFGF or passage, and having shown does not have macroscopical host tissue to invade.Fig. 5 B shows the PEG hydrogel of being gathered in the crops with 3 major paths (being diameter 1mm), and it shows host tissue inwardly growth in the chamber of engineering major path.Fig. 5 C shows the load bFGF gathered in the crops but does not have the PEG hydrogel of major path that it shows that integral body is redness.Fig. 5 D show gathered in the crops have bFGF and major path and this PEG hydrogel, it shows that integral body be redness, and host tissue is inwardly grown in the chamber of 3 engineering major paths.Ratio scale: 6mm.From people such as Stosich (2006).Further details about this method is provided in embodiment 3.

Fig. 6 is a series of figure, and it is presented at body and is implanted into 3 weeks back use H﹠amp; The painted PEG hydrogel sample of E.Fig. 6 A representative does not have bFGF and major path PEG hydrogel (H), and its demonstration does not have host cell to invade.Fig. 6 B represents host tissue, and it is grown in the PEG hydrogel (H) with 3 major paths (C arrow).Attention part beyond the major path in PEG does not have the host cell infiltration.Fig. 6 C has described load bFGF but has not had the PEG hydrogel (H) of passage, and it shows obviously host tissue infiltration at random.Fig. 6 D represents the host tissue infiltration; This infiltration occurs over just in the major path of the PEG hydrogel that bFGF-soaks into.Although Fig. 6 C is identical with the dosage of bFGF among Fig. 6 D, the PEG (Fig. 6 D) with load bFGF of major path has induced a large amount of host cells inwardly to grow.From people such as Stosich (2006).Further details about this method is provided in embodiment 3.

Fig. 7 is a block diagram, and it shows the amount by the ingrown host tissue of computer organization norphometry.The amount of ingrown host tissue is bigger significantly than the amount of host tissue in the major path of the PEG hydrogel that does not have bFGF in the major path of the PEG of load bFGF hydrogel.Every group of N=8.From people such as Stosich (2006).Further details about this method is provided in embodiment 3.

Fig. 8 is an a series of pictures, and it is depicted in the H﹠amp of ingrown host tissue in the PEG hydrogel; E dyeing.Fig. 8 A representative has major path, but does not have the PEG hydrogel of bFGG, and it shows only inwardly growth in major path of host tissue.Arrow is represented blood vessel.Fig. 8 B represents more high-power Fig. 8 A, and it shows the capillary structure of being lined up by endotheliocyte class cell (white arrow), and is centered on by fibroblast class cell.Fig. 8 C represents PEG hydrogel (H), load bFGF but do not have major path, and it shows seldom ingrown host tissue, the capillary structure of being lined up by endotheliocyte class cell (black arrow).Fig. 8 D represents more high-power Fig. 8 C.Fig. 8 E representative has the PEG hydrogel of bFGF and major path, and it shows with the ingrown tight host tissue of high density capillary structure (black arrow).The more high power picture of Fig. 8 F representative graph 8E, its demonstration have the trunk spline structure (white arrow) of similar erythrocyte, and are lined up by endotheliocyte class cell.Fibroblast class cell is around capillary structure.From people such as Stosich (2006).Further details about this method is provided in embodiment 3.

Fig. 9 is a series of figure, and it describes to use the painted immunolocalization tissue slice of anti-VEGF antibodies.Fig. 9 A represents PEG hydrogel (H), and it does not have bFGF and major path, and it shows that lacking the VEGF positive except host's fibrous capsule (C) organizes.Fig. 9 B represents the PEG hydrogel, has 3 major paths but does not have bFGF, and it shows strong VEGF dyeing of host tissue in major path.Fig. 9 C represents PEG hydrogel (H), load bFGF but do not have major path, and it shows with obvious at random the positive tissue of VEGF of mode.Fig. 9 D represents PEG hydrogel (H), has bFGF and major path, and it is presented at the strong VEGF dyeing of host tissue in the major path.From people such as Stosich (2006).Further details about this method is provided in embodiment 3.

Figure 10 is a series of sketches, and it describes the experimental establishment of cell density experiment.Human mesenchymal stem cell (MSC), MSC-are derivatized to osteocyte (MSC-Ob) and the MSC chondrocyte (MSC-Cy) of deriving.For every kind of cell lineage, four kinds of cell densities of encapsulation in the PEG hydrogel: 0,5,000,000,4,000 ten thousand and 8,000 ten thousand cells of every ml cells suspension.The OS culture medium: osteogenesis stimulates culture medium, and it contains dexamethasone (dexamethosone), ascorbic acid and b-phosphoglycerol.CS culture medium cartilage generates culture medium, and it contains TGFb3.Figure 10 A representative MSC is not divided into any cell lineage.The deutero-osteoblast of Figure 10 B representative MSC.The deutero-chondrocyte of Figure 10 C representative MSC.In each case, with the 3D cultured cells by trypsin treatment and be loaded in the cell suspending liquid.Then the cell that suspends is loaded into the aqueous phase of PEG hydrogel, then carries out photopolymerization and form gel.For every kind of condition (A, B and C), the gelation construct that has obtained encapsulation MSC, MSC-Ob and MSC-Cy is used for research in further external and the body.From Troken and Mao (2006).Further details about this method is provided in embodiment 4.

Figure 11 is a series of figure, and it is depicted in In vitro culture after 4 weeks, the structure observation of various cell densities.Last row: the contrast or the MSC that do not have differentiation that in DMEM, cultivate.Middle row: the MSC-osteoblast of in the skeletonization culture medium, cultivating (MSC-Ob).Following row: the deutero-chondrocyte of in becoming the cartilage culture medium, cultivating of MSC (MSC-Cy).5,000,000 cells of the every ml cells suspension of 5M cell/mL=.Left column is represented acellular PEG hydrogel.The initiator cell inoculum density of every milliliter of 5,000,000 cells of next column representative then is every milliliter of 4,000 ten thousand cells, and right column is every milliliter of 8,000 ten thousand cells.For every kind of cell lineage, in incubated in vitro, keep initiator cell 4 weeks of inoculum density.From Troken and Mao (2006).Further details about this method is provided in embodiment 4.

Figure 12 is a series of figure, and it describes to be depicted in the safranin O dyeing of In vitro culture PEG hydrogel after 4 weeks, wherein PEG hydrogel encapsulation human mesenchymal stem cell (MSC) (Figure 13 A-13D) and the MSC chondrocyte (MSC-Cy) (Figure 13 A '-13D ') of deriving.Left column is represented acellular PEG hydrogel.The initiator cell inoculum density of every milliliter of 5,000,000 cells of next column representative then is every milliliter of 5,000 ten thousand cells, and right column is every milliliter of 8,000 ten thousand cells.Male safranin O dyeing has shown the labelling area relevant with the initiator cell inoculum density.MSC is that safranin O dyeing is negative.The initiator cell inoculum density is kept together with the one-tenth cartilage phenotype that breaks up in the PEG hydrogel.From Troken and Mao (2006).Further details about this method is provided in embodiment 4.

Figure 13 is a series of figure, and it is depicted in the Feng Kusa dyeing of In vitro culture PEG hydrogel after 4 weeks, and wherein PEG hydrogel encapsulation human mesenchymal stem cell (MSC) (Figure 14 A-14D) and MSC are derivatized to osteocyte (MSC-Ob) (Figure 14 A '-14D ').Left column is represented acellular PEG hydrogel.The initiator cell inoculum density of every milliliter of 5,000,000 cells of next column representative then is every milliliter of 4,000 ten thousand cells, and right column is every milliliter of 8,000 ten thousand cells.Feng Kusa dyeing is positive, and has shown the labelling area relevant with the initiator cell inoculum density.MSC is that Feng Kusa dyeing is negative.When this hinted the skeletonization stimulus object of row under not having adding, MSC was not divided into osteoblast.The initiator cell packaging density is kept together with the one-tenth bone phenotype that breaks up in the PEG hydrogel.From Troken and Mao (2006).Further details about this method is provided in embodiment 4.

Figure 14 is pair of columnar figure, and it shows derives MSC to be derivatized to osteocyte substrate formed quantitatively for chondrocyte and MSC.Figure 14 A representative 4 week body account for the Alcian blueness gross area of entire bracket area after being implanted into.The MSC chondrocyte (MSC-Cy) of deriving has synthesized than hMSC and HMSC and has been derivatized to the GAG that osteocyte (hMSC-Ob) is Duoed.Figure 14 B representative accounts for the Feng Kusa dyeing gross area of entire bracket area.The mineralising that MSC-Ob induction ratio hMSC and HMSC-Cy are significantly many.Every group of N=8.From Troken and Mao (2006).Further details about this method is provided in embodiment 4.

Figure 15 is a series of sketches, and it describes the structure of PEG hydrogel and the immunohistochemistry figure of the hydrogel of implanting after corresponding 4 weeks.Figure 15 A describes the PEG hydrogel, has major path but does not have bFGF.Figure 15 B describes the PEG hydrogel, has bFGF but does not have major path.Figure 15 C describes the PEG hydrogel, has major path and bFGF.Figure 15 A ' is that the immunohistochemistry of the PEG hydrogel of Figure 15 A that implants is organized picture.Figure 15 B ' is that the immunohistochemistry of the PEG hydrogel of Figure 15 B that implants is organized picture.Figure 15 C ' is that the immunohistochemistry of the PEG hydrogel of Figure 15 C that implants is organized picture.Further details about this method is provided in embodiment 20.

Figure 16 is a series of figure, and it is described human mesenchymal stem cell and became give birth to lipocyte in vitro differentiation at 35 days in the isolated culture process.Use oil red O stain, the deutero-living lipocyte of hMSC and its reacting positive.The hMSC of lipocyte differentiation is not given birth in Figure 16 A-16E representative, and Figure 16 F-16J represents the deutero-living lipocyte of hMSC.Further details about this method is provided in embodiment 21-22.

Figure 17 is a series of block diagrams, and it shows the total dna content (Figure 17 A) through 35 days culture samples between hMSC and the deutero-living lipocyte of hMSC, and the glycerol content (Figure 17 B) of hMSC and the deutero-living lipocyte sample of hMSC.Further details about this method is provided in embodiment 22.

Figure 18 is a series of sketches and picture, and its hMSC and vascularization after implanting for 4 weeks of the deutero-living lipocyte of hMSC of describing to be encapsulated in the PEG hydrogel generates fat.Figure 18 A describes the PEG hydrogel, does not have major path, does not have bFGF, does not have cell to send.Figure 18 B describes the PEG hydrogel, has major path, has bFGF, but does not have cell to send.Figure 18 C describes the PEG hydrogel, and it has major path, has bFGF, and sends the hMSC-adipose cell.Figure 19 A ', 19B ' and 19C ' are respectively that the PEG hydrogel of Figure 19 A, 19B and 19C is being implanted the photo of mice after 12 weeks.Further details about this method is provided in embodiment 23.

Figure 19 is a series of figure, and it describes dyed tissue slice, wherein encapsulates implanted 12 week of PEG microchannel hydrogel with major path of the deutero-living lipocyte of hMSC.Figure 19 A is the tissue of immunohistochemical staining.Figure 19 B is by the tissue of oil red O (Oil-red O) stained positive.Figure 19 C is by the painted tissue of VEGF antibody.Figure 19 D is by the tissue of anti-WGA lectins antibody staining.Further details about this method is provided in embodiment 23.

Figure 20 is a series of figure, and it shows the expression of blood vessel CFU-GM medium vessels endothelial cell growth factor (ECGF) 2 or Flk1.Further details about this method is provided in embodiment 24.

Figure 21 is a block diagram, and VEGF2's is quantitative in its demonstration blood vessel CFU-GM.Further details about this method is provided in embodiment 24.

Figure 22 is a picture, and it is presented in the β TCP support by the osteoprogenitor cells of green fluorescent protein (GFP) labelling with by the blood vessel CFU-GM of red CM-DiI labelling.Further details about this method is provided in embodiment 25.

Detailed Description Of The Invention

Approach described herein is at least part of based on passing through to use candidate stem cell and mesenchyma The synergy of stem cell forms the discovery of blood vessel tissue to carry out organizational project. This paper demonstrate,proves Bright is when organizing the combination of CFU-GM and blood vessel CFU-GM, the blood vessel of polymeric biomaterial Change. Also proved in being introduced in the porous support that contains CFU-GM in a organized way or on it the time, blood The pipe CFU-GM is the induction of vascular spline structure in vivo. Further proved physics in host material Built-in major path and/or angiogenesis growth factor are induced the Angiogenesis of host derivation in vivo And vascularization.

Therefore, provide a kind of by the blood vessel CFU-GM with organize the two synergy of CFU-GM Remedy the New Regenerated approach of tissue defects, so as total effect can greater than effect separately and. These approach are benefited from disclosed herein about blood in from the beginning forming blood vessel tissue or organ Manage CFU-GM (for example HSC), organize CFU-GM (for example MSC) and their cell spectrum Be that derivative and modulating vascular generate the interactional new understanding between the growth factor. As example Son, composition disclosed herein and method can provide the upward great-hearted engineering of biology sclerous tissues Assembly is used for repairing for example segmental defective of long bone defective, in biologically-derived total joint replacement Subchondral bone regeneration, and marrow displacement. As another example, described herein group Compound and method can provide biological great-hearted engineering soft fat tissue, be used for repairing owing to The caused soft tissue defective of wound, tumorectomy and birth defect.

One aspect of the present invention provides the composition of engineering blood vessel tissue or organ. These Composition generally includes and is introduced in the biocompatible matrix or organizes CFU-GM and blood on it The pipe CFU-GM. Another aspect of the present invention provides and has been used to form these engineering blood vessel tissues Or the method for organ. According to these methods that is used for organizational project and regeneration, organize the ancestral thin Born of the same parents and blood vessel CFU-GM are introduced in the biocompatible matrix or on it, to produce the vascularization group Knit or organ. Further the aspect provides by composition of the present invention being transplanted to this needs Come the method for treated tissue defective in the individuality of wanting.

Great-hearted tissue or organ can be from having the CFU-GM of organizing that improves vascularization on the biology Built by using the blood vessel CFU-GM. The blood that can form according to method disclosed herein Pipeization tissue or organ type include but not limited to bladder, bone, and brain, breast, the bone cartilage connects The place, nerve fiber comprises central nervous system, spinal cord and peripheral nerve, neuroglia, esophagus, Fallopian tubal, heart, pancreas, intestines, gall-bladder, kidney, liver, lung, ovary, prostate, spinal cord, Spleen, skeletal muscle, skin, stomach, testis, thymus gland, thyroid gland, tracheae, urogenital tract, Ureter, urethra, a matter soft tissue, periosteum, periodontium, sutura cranii, hair follicle, the oral cavity is sticking Film and uterus. The engineering blood vessel by the formed preferred soft tissue composition of method of the present invention Change adipose tissue. Engineering blood by the formed preferred sclerous tissues of method of the present invention composition The pipeization bone tissue.

Organize the aggregate of the cell that normally has similar morphology and function, and usually obtain Support with allos interstitial tissue of various kinds of cell type and blood supply. Usually organ is to send out Wave the aggregate of the tissue of biological function. Organ can be but be not limited to bladder, brain, neural group Knit, neuroglia tissue, esophagus, fallopian tubal, bone, synovial joint (synovial joint), Sutura cranii, heart, pancreas, intestines, gall-bladder, kidney, liver, lung, ovary, prostate, spinal cord, Spleen, stomach, testis, thymus gland, thyroid gland, tracheae, urogenital tract, ureter, urethra, Uterus, breast, skeletal muscle, skin, bone and cartilage. Can use those skilled in the art The biological function of the standard method check organ of knowing.

Infusion and cultivation

In order to form composition of the present invention, will organize CFU-GM and blood vessel CFU-GM to introduce (example Such as implantation, injection, infusion or inoculation) artificial to supporting that three-dimensional tissue or organ form In the structure (support that for example comprises host material) or on it. Can CFU-GM and blood will be organized The pipe CFU-GM is introduced simultaneously or successively. Can will organize CFU-GM to be incorporated into mutually with the blood vessel CFU-GM Identical locus mutually, similarly locus or different locus. Preferably, Organize CFU-GM and blood vessel CFU-GM to be introduced in the zones of different of host material or on it. In advance Phase can be incorporated into polytype CFU-GM of organizing in the matrix. Similarly, expection can with Polytype blood vessel CFU-GM is incorporated in the matrix.

Can will organize CFU-GM and/or blood vessel CFU-GM to draw by multiple means known in the art Enter in host material (referring to for example embodiment 1; Embodiment 4; Embodiment 11; Embodiment 12; Embodiment 20, and embodiment 23). Be used for CFU-GM is introduced (for example infusion, inoculation, Injection etc.) in the host material or the method in it at for example Ma and Elisseeff, ed. (2005) Scaffolding In Tissue Engineering, CRC ISBN 1574445219; Saltzman (2004) Tissue Engineering:Engineering Principles for the Design Of Replacement Organs and Tissues, Oxford ISBN 019514130X; Minuth Deng people (2005) Tissue Engineering:From Cell Biology to Artificial Organs, John Wiley ﹠ Sons comes into question among the ISBN 3527311866. For example, have by comprising (for example, concentration is the hydration of 100 cell/ml～millions of cell/ml) to cell suspending liquid The method of freeze-drying support, CFU-GM be directed in the matrix or on it. Add additional agents Method can change as discussed below.

Cultivate and differentiation support (scaffold) in or the CFU-GM on the support be known in the art (referring to for example Saltzman (2004) Tissue Engineering:Engineering Principles For the Design of Replacement Organs and Tissues, Oxford ISBN 019514130X; Vunjak-Novakovic and Freshney, eds. (2006) Culture of Cells For Tissue Engineering, Wiley-Liss, ISBN 0471629359; The people such as Minuth (2005) Tissue Engineering:From Cell Biology to Artificial Organs, John Wiley ﹠ Sons, ISBN 3527311866). Just as skilled in the art will understand, the ancestral is thin Born of the same parents be incorporated in the matrix or it on and the time between the resulting matrix of transplanting can be according to specific Application and change. Hatch (and subsequent duplicate and/or differentiation) in host material or on it Containing in a organized way, the engineering composition of CFU-GM and blood vessel CFU-GM can be for example at least partially in body Outward, basically external, in vivo at least part of, perhaps basically in vivo. Determine best Incubation time belong to the technical ability of this area. It is thin to use suitable culture medium to be used for external ancestral Born of the same parents' infusion, differentiation or Cell Differentiation transfer (referring to for example Vunjak-Novakovic and Freshney, Eds. (2006) Culture of Cells for Tissue Engineering, Wiley-Liss, ISBN 0471629359; The people such as Minuth (2005) Tissue Engineering:From Cell Biology To Artificial Organs, John Wiley ﹠ Sons, ISBN 3527311866). Incubation time Can in about 1 hour, several hours, one day, several days, a week or a few week, change. Morphology that can be by for example utilizing ELISA, by the protein check, by the heredity check, By mechanical analysis, by RT-PCR and/or by immunostaining screening cell type specificity Label comes the amount of the cell that exists in the matrix and type are identified (referring to for example Minuth Deng people (2005) Tissue Engineering:From Cell Biology to Artificial Organs, John Wiley ﹠ Sons, ISBN 3527311866).

As described herein, in some embodiments, by will organize CFU-GM and The blood vessel CFU-GM is incorporated in the host material or on it, forms engineering blood vessel tissue or organ, And do not need to use extra bioactivator, particularly growth factor etc. Do not exist growth because of The ability that the period of the day from 11 p.m. to 1 a.m forms engineering blood vessel tissue provides traditional handicraft institute's tool not in organizational project The advantage that has.

Vascularization

Under the condition that causes the composition vascularization, generation will be organized CFU-GM and blood vessel CFU-GM Be incorporated in the host material or on it. Preferably, vascular tissue is in whole engineered tissue or organ Growth. Can be external (referring to for example embodiment 2; Embodiment 22), in the body (referring to for example Embodiment 1; Embodiment 3) or its combination, in organizational project or organ, produce vascularization. Example As, can be undertaken by cultivating intracellular CFU-GM and the blood vessel CFU-GM organized of backing substrate Differentiation. As another example, CFU-GM can be infused in the matrix, and rapidly with this Matrix is transplanted in the individuality, so that break up in vivo. When engineered tissue or organ are drawn Enter the individual amount of determining to be based, at least in part, on the vascularization that forms in tissue or the organ.

The method that is used for the generation of measurement organizational project or organ medium vessels is this area Plays (referring to such as the people such as Jain (2002) Nat.Rev.Cancer 2:266-276; Ferrara, Ed. (2006) Angiogenesis, CRC, ISBN 0849328446). In early days vascularization In the journey, in growth course, jejune blood vessel is by having relatively big diameter and lacking blood Tubular attitude differentiation and be similar to plexus vasculosus. Along with the past of time, jejune Angiogenesis blood The mesh mode of pipe gradually maturation is functional microcirculation unit, and it develops into has the thin of differentiation Artery and venular intensive capillary network. Can be by for example measuring no branch vessel sections Number (number of per unit area inner segments), (the per unit area perfusion of functional vessel density The total length of blood vessel), blood vessel diameter or blood vessel bulk density are (according to the length of each sections and straight Calculate in the footpath, per unit area blood vessel cumulative volume).

Compositions of the present invention has the beguine vascularization that engineered tissue that means produced or organ improve of reportedly uniting usually.For example, with be not by introducing blood vessel CFU-GM described herein with organize the two formed corresponding engineered tissue of CFU-GM or organ to compare, the vascularization in engineered tissue or the organ (for example the foundation of jejune blood vessel network, blood vessel remodeling, blood vessel stabilisation, mature blood vesselization, blood vessel differentiation or functional blood vessel network takes place, forms for angiogenesis, blood vessel) can be enhanced at least 5%, 10%, 20%, 25%, 30%, 40% or 50%, 60%, 70%, 80%, 90% even reach 100%, 150% or 200%.Preferably, the vascularization of engineered tissue or organ compositions is stable blood vessel network, and can there be at least 1 day in it, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or even 12 months or longer.Preferably, the blood vessel network of engineered tissue or organ compositions is incorporated into tissue, organ or individual blood circulation by introducing.

For tissue or the neomorph of using small rack (size is less than 100 cubic millimeters), can external manual change culture medium, and regularly (for example per 3～4 days) add additional agents.For big support, cultivation can be remained in the biological example reactor assembly, described bioreactor system can use micropump to be used to change culture medium.Micropump can be arranged in the couveuse, with the host material of fresh culture medium pump to support.The culture medium circulation is reclaimed, and by like this, substrate can have about fresh culture of 1%～about 100%.Pump speed can be conditioned the optimal allocation that is comprised additional agents in culture medium and/or the culture medium to reach.Can be according to the tissue of manufacturing or the type customization culture medium delivery system of organ.Preferably, under aseptic condition, carry out all cultivations.

CFU-GM

The compositions and methods of the invention adopt organize CFU-GM and blood vessel CFU-GM the two.These cells can be by the whole bag of tricks separation as known in the art, purification and/or cultivation (referring to for example embodiment 9; Embodiment 21).The method that is used to separate and cultivates CFU-GM comes into question at for example Vunjak-Novakovic and Freshney (2006) Culture of Cells for TissueEngineering, and Wiley-Liss is among the ISBN 0471629359.In certain aspects, CFU-GM can be derived from the species identical or different with the receptor that is intended to transplant.For example, CFU-GM can include but not limited to vertebrates for example mammal, reptile or bird derived from animal.In some configuration, preferred mammal or birds are horse, cattle, Canis familiaris L., cat, sheep, pig or chicken, and optimum is chosen.

Of the present inventionly organize CFU-GM to comprise to be divided into destination organization or organ and/or experience form to form to form the cell of destination organization or organ.Organize the non-limitative example of CFU-GM to comprise mescenchymal stem cell (MSC), cell from the MSC differentiation, osteoblast, chondrocyte, the myocyte, adipose cell, neuronal cell, the neuron sustenticular cell is neurogliocyte (for example Schwann cell) for example, the fibroblast cell for example between the matter fibroblast, tendon fibroblast (tendon fibroblast), dermal fibroblast, the ligament fibroblast, the periodontal fibroblast is the gums fibroblast for example, cranium face fibroblast, myocardial cell, epithelial cell, hepatocyte, the urethra cell, nephrocyte, periosteum cell, the bladder cell, β-islet cells, odontoblast, pulp cells, periodontal cell, pneumonocyte and heart cell.For example, in vascularization osseous tissue of the present invention, the CFU-GM of organizing that is incorporated in the substrate can be CFU-GM for example mescenchymal stem cell (MSC), MSC osteoblast or the MSC chondrocyte that can produce osseous tissue.Need to understand, the MSC chondrocyte is the chondrocyte from the MSC differentiation.Similarly, the MSC osteoblast is an osteoblast MSC osteoblast.In another example, in the Vascularized fat depot of the present invention, the CFU-GM of organizing that is incorporated in the substrate can be the CFU-GM that can produce fatty tissue, and for example MSC or MSC give birth to lipocyte (i.e. the living lipocyte that breaks up from MSC).

Be incorporated in the host material or the blood vessel CFU-GM on it is the CFU-GM that can be divided into or otherwise form vascular tissue.The blood vessel CFU-GM can be the stem cell hematopoietic stem cell (HSC) for example that for example can be divided into endotheliocyte, the HSC endotheliocyte, vascular endothelial cell, lymphatic endothelial cells, the endotheliocyte pedigree, the former foster endotheliocyte of being commissioned to train, endotheliocyte derived from stem cell, the bone marrow derived stem cell, notochord blood derived cell, Human umbilical vein endothelial cells (HUVEC), the lymph gland endotheliocyte, endothelial progenitor cells, the endotheliocyte pedigree, the external endotheliocyte that produces from stem cell, endotheliocyte from the fatty tissue extraction, smooth muscle cell, between the matter fibroblast, myofibroblast, periodontal tissue, dental pulp or blood vessel derived cell.Need to understand, the HSC endotheliocyte is the endotheliocyte from the HSC differentiation.Can be from for example bone marrow, soft tissue, muscle, blood and/or vascular system separating blood vessel CFU-GM.In some configuration, the blood vessel CFU-GM can be derived from organizing CFU-GM.

The present invention includes to optimize and organize the density of CFU-GM and blood vessel CFU-GM the two (and their pedigree derivant) so that make the maximized method of regeneration result of blood vessel tissue or organ (referring to for example embodiment 4; Embodiment 5; Embodiment 6).In these methods, can monitor with in the substrate through after a while and at the cell density of terminal point.Can determine the character organized, for example use standard technique well known by persons skilled in the art, for example histology, structural analysis, immunohistochemistry, biochemical analysis and mechanical performance.Can recognize as those skilled in the art, organize CFU-GM and/or the blood vessel progenitor cells density can be according to for example CFU-GM type, tissue or organ type, host material, substrate volume, infusion methods, inoculation pattern, culture medium, somatomedin, incubation time, incubation conditions etc. change.Usually, for organize CFU-GM and blood vessel CFU-GM the two, the cell density of every kind of cell can be 0.0001 * 10 independently in the substrate ⁶Individual cell (M) ml ^-1～about 1000Mml ^-1For example, organize CFU-GM and blood vessel CFU-GM all can in substrate, exist: about 0.001Mml with following density ^-1, 0.01Mml ^-1, 0.1Mml ^-1, 1Mml ^-1, 5Mml ^-1, 10Mml ^-1, 15Mml ^-1, 20Mml ^-1, 25Mml ^-1, 30Mml 1,35Mml ^-1, 40Mml ^-1, 45Mml ^-1, 50Mml ^-1, 55Mml ^-1, 60Mml ^-1, 65Mml ^-1, 70Mml ^-1, 75Mml ^-1, 80Mml ^-1, 85Mml ^-1, 90Mml ^-1, 95Mml ^-1, 100Mml ^-1, 200Mml ^-1, 300Mml ^-1, 400Mml ^-1, 500Mml ^-1, 600Mml ^-1, 700Mml ^-1, 800Mml ^-1Or 900Mml ^-1

Can be with various ratios with blood vessel CFU-GM and organize CFU-GM to be incorporated in the substrate or (referring to embodiment 5) on it.Just as skilled in the art will understand, the blood vessel CFU-GM with organize the progenitor cells ratio to change according to for example CFU-GM type, destination organization or organ type, host material, substrate volume, infusion methods, inoculation pattern, culture medium, somatomedin, incubation time and/or incubation conditions etc.Usually, the blood vessel CFU-GM can be about 100:1～about 1:100 with the ratio of organizing CFU-GM.For example the blood vessel CFU-GM can be about 20:1,19:1,18:1,17:1,16:1,15:1,14:1,13:1,12:1,11:1,10:1,9:1,8:1,7:1,6:1,5:1,4:1,3:1,2:1,1:1,1:2,1:3,1:4,1:5,1:6,1:7,1:8,1:9,1:10,1:11,1:12,1:13,1:14,1:15,1:16,1:17,1:18,1:19 or 1:20 with organizing the ratio of CFU-GM.

In some embodiments, the CFU-GM that is introduced into substrate can comprise heterologous nucleic acids so that express for example heterologous protein or be used for expressing heterologous albumen of bioactive molecule.In nonrestrictive example, the CFU-GM that is introduced into substrate can express fluorescent protein label, for example GFP, EGFP, BFP, CFP, YFP or RFP.In another example, the CFU-GM that is introduced into substrate can be expressed angiogenesis correlation factor for example activin A, adrenomedullin, aFGF, ALK1, ALK5, ANF, angiogenin, angiopoietin-1, vascularization element-2, vascularization element-3, vascularization element-4, angiostatin, blood vessel attraxin, angiotensin-2, AtT20-ECGF, β cytokines, bFGF, B61, bFGF induced activity, cadherins, CAM-RF, cGMP analog, ChDI, CLAF, claudins, collagen, collagen receptor α ₁β ₁And α ₂β ₁Connect albumen, Cox-2, ECDGF (endotheliocyte derivative growth factor), ECG, ECI, EDM, EGF, EMAP, endoglin, Endothelin, endostatin, the endothelial cell growth inhibitor, the endotheliocyte vigor is kept the factor, endothelium differentiation sheath sugar g protein coupled receptor-1 (EDG1), ephrins, Epo, HGF, TNF-α, TGF-β, PD-ECGF, PDGF, IGF, IL8, growth hormone, fibrin fragment E, FGF-5, fibronectin and fibronectin receptor α ₅β ₁, factor X, HB-EGF, HBNF, HGF, HUAF, vascular cell amplification cardiac-derived inhibitor, IFN-γ, IL1, IGF-2IFN-γ, integrin receptor (α subunit (α for example for example ₁, α ₂, α ₃, α ₄, α ₅, α ₆, α ₇, α ₈, α ₉, α _E, α _V, α _IIb, α _L, α _M, α _X) various combinations, K-FGF, LIF, the leiomyoma derivative growth factor, MCP-1, macrophage derived growth factor, the monocyte derived somatomedin, MD-ECI, MECIF, mmP2, mmP3, mmP9, the urokinase plasminogen activators, neuropil albumen (NRP1, NRP2), neurothelin, nitric oxide donors, nitric oxide synthetase (NOS), notch, closed albumen, banded closed albumen, oncostatin M, PDGF, PDGF-B, pdgf receptor, PDGFR-β, PD-ECGF, PAI-2, PD-ECGF, PF4, P1GF, PKR1, PKR2, PPAR-γ, PPAR-γ part, phosphodiesterase, prolactin antagonist, prostacyclin, protein S, the smooth muscle cell derivative growth factor, the smooth muscle cell migration factor of deriving, sphingosine-1-phosphate-1 (S1P1), Syk, SLP76, tachykinin, TGF-β, Tie1, Tie2, TGF-β and TGF-beta receptor, TIMP, TNF-α, TNF-β, transferrins, thrombospondin, urokinase, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF, VEGF ₁₆₄, VEGI, EG-VEGF, vegf receptor, PF4, prolactin antagonist 16kDa fragment, PGE1 and E2, steroid, heparin, 1-butyryl glycerol (only son's acid glyceride) or niacin amide.As another example, the CFU-GM that is introduced into substrate can contain genetic sequence, its reduction or eliminate immunne response among the host (for example for example I class and II class are organized compatible antigenic expression by suppressing cell surface antigen).

In some embodiments, organize the CFU-GM and the first blood vessel CFU-GM except first, can also be incorporated into one or more cell types in the host material or on it.These extra cell types can be selected from discussed above, and/or can include, but is not limited to Skin Cell, hepatocyte, heart cell, nephrocyte, pancreatic cell, pneumonocyte, bladder cell, gastric cells, enterocyte, urogenital tract cell, mammary cell, Skeletal Muscle Cell, Skin Cell, osteocyte, chondrocyte, keratinocyte, hepatocyte, gastrointestinal cell, epithelial cell, endotheliocyte, mammary glandular cell, Skeletal Muscle Cell, smooth muscle cell, soft tissue cells, osteoclast or chondrocyte.Can before the substrate vascularization, in the process, or introduce these cell types afterwards.This introducing can occur in external or the body.If introduce these cells in vivo, then introducing can be in the site of engineering blood vessel tissue or organ compositions, perhaps with its irrelevant site.The exemplary route of using these cells comprises injection and operation implantation.

Substrate

The compositions and methods of the invention adopt substrate, to introducing CFU-GM wherein or it on, so that formative tissue or organ construct.These host materials can allow cell attachment and migration; Send and keep the cell and the biochemical factor; Realize the diffusion of cytotrophy thing and expressed product; And/or bring into play some machinery and biological impact with the change cell stage behavior.Usually, substrate is the porous micropore support of bio-compatible material, and it provides physical support and adhere to base material and is used for being implanted into process introducing blood vessel CFU-GM and organizing CFU-GM at In vitro culture and follow-up body.Preferably, has the substrate of highly porous and suitable pore size, to promote introducing cell and diffusion cell and nutrient in total.The biodegradability of substrate also is preferred, because surrounding tissue can be avoided needs that operation is removed to the absorption of substrate.The speed that degraded takes place need be consistent as far as possible with the speed that tissue or organ form.Therefore, when cell at construct during around their self natural structure, substrate can provide the globality of structure, and finally decomposes and stay cambium, new tissue or the organ that forms, it can present mechanical load.Syringeability also is preferred in some clinical practice.Suitable host material is described in for example Ma and Elisseeff, ed. (2005) Scaffolding in Tissue Engineering, CRC, ISBN1574445219; Saltzman (2004) Tissue Engineering:Engineering Principlesfor the Design of Replacement Organs and Tissues is among the Oxford ISBN019514130X.

Matrix structure can since to be repaired or the tissue or the organ that produce, but preferred substrate is a foraminous die plate softish, bio-compatible, it allows blood vessel and destination organization or organ growth.Substrate can be manufactured into the structural support thing, and wherein the layout of this structure (for example shape, size, porous, microchannel or major path) is customized according to application.The porous of substrate is a kind of design parameter, and it influences cell and introduces and/or Premeabilisation of cells.Substrate can be designed to adopt the extracellular matrix proteins that influences cell adhesion and move in substrate.

Substrate can be formed by synthetic polymer.These synthetic polymers include but not limited to polyurethane, poe, polyvinyl alcohol, polyamide, Merlon, Polyethylene Glycol, polylactic acid, polyglycolic acid, polyvinylpyrrolidone, ocean attachment proteins (marine adhesive protein) and cyanoacrylate or analog or above-mentioned mixture, combination and derivant.

Interchangeable, substrate can be formed by naturally occurring polymer or natural derived polymers.These polymer include but not limited to agarose, alginate, fibrin, Fibrinogen, fibronectin, collagen, gel, hyaluronic acid and other suitable polymer and biopolymer or above-mentioned analog, mixture, combination and derivant.Equally, substrate can form from the mixture of naturally occurring biopolymer and synthetic polymer.

Host material substrate can comprise for example collagen gel, polyvinylalcohol sponge, poly-(D, L-lactide-copolymerization-Acetic acid, hydroxy-, bimol. cyclic ester) fibre substrate, poly lactose (polyglactin) fiber, calcium alginate gel, polyglycolic acid net, polyester (for example poly-(L-lactic acid) or polyanhydride), polysaccharide (for example alginate), polyphosphazene or polyacrylate or poly(ethylene oxide)-polypropylene glycol block copolymer.Substrate can form from protein (for example extracellular matrix protein for example fibrin, collagen and fibronectin), polymer (for example polyvinylpyrrolidone) or hyaluronic acid.Also can use synthetic polymer, it comprises biological erodable polymer (for example poly-(lactide); poly-(glycolic); poly-(lactide-copolymerization-Acetic acid, hydroxy-, bimol. cyclic ester); poly-(caprolactone); Merlon; polyamide; polyanhydride; polyamino acid; poe; polyacetals; polybutylcyanoacrylate); degradable polyurethane; not erodible polymer (polyacrylate for example; the cellulose acetate of ethylene-vinyl acetate polymer and other acyl substituted and its derivant); not erodible polyurethane; polystyrene; polrvinyl chloride; polyvinyl fluoride; poly-(vinyl imidazole); the chloro sulfonated polyolefin; poly(ethylene oxide); polyvinyl alcohol; teflon

And nylon.

Substrate can also contain one or more of enzyme, ion, somatomedin and/or biological reagent.For example substrate can contain somatomedin (for example angiogenesis growth factor or tissue specificity somatomedin).This class somatomedin can be provided with the concentration of about 0～1000ng/mL.For example somatomedin can be with the concentration of about 100～700ng/mL, and with the concentration of about 200～400ng/mL, perhaps the concentration with about 250ng/mL exists.

Substrate can contain one or more physical channel.These physical channels comprise microchannel and major path.The microchannel has the diameter of about 0.1 μ m～about 1,000 μ m usually.Shown as this paper, the substrate major path can promote angiogenesis and bone or fatty tissue to form, and the generation of guiding vascularization and host cell invasion is (referring to for example embodiment 3; Embodiment 20; Embodiment 23).Microchannel and/or major path can be the parts of special construct in naturally occurring parts of some host material and/or the host material.The formation of microchannel and/or major path can be according to for example machinery and/or chemical means.

Major path can extend the multiple degree of depth in the substrate, perhaps passes completely through substrate.Major path can be a multiple diameter.Usually can select the diameter of major path according to the vascularization of the perfusion optimization, tissue growth and the organization component that improve.Major path for example can have the approximately average diameter of 0.1mm～about 50mm.For example, major path can have following average diameter: about 0.2mm, about 0.3mm, about 0.4mm, about 0.5mm, about 0.6mm, about 0.7mm, about 0.8mm, about 0.9mm, about 1.0mm, about 1.1mm, about 1.2mm, about 1.3mm, about 1.4mm, about 1.5mm, about 1.6mm, about 1.7mm, about 1.8mm, about 1.9mm, about 2.0mm, about 2.5mm, about 3.0mm, about 3.5mm, about 4.0mm, about 4.5mm, about 5.0mm, about 5.5mm, about 6.0mm, about 6.5mm, about 7.0mm, about 7.5mm, about 8.0mm, about 8.5mm, about 9.0mm, about 9.5mm, about 10mm, about 15mm, about 20mm, about 25mm, about 30mm, about 35mm, approximately 40mm or approximately 45mm.

Ability technical staff can understand, and the distribution of major path diameter can be the normal state diameter Distribution, perhaps the abnormal diameter Distribution.

The medicine and/or the diagnostic agent that add

In some embodiments, method and composition of the present invention also comprises other reagent, together with organizing CFU-GM and blood vessel CFU-GM to be introduced in the substrate or on it.Various actual bioactive molecule, bio-pharmaceutical, diagnostic reagent and the reinforcing agent of including but not limited to that can introduce.

Substrate can also contain bioactive molecule.The cell of substrate can perhaps can join bioactive molecule in the substrate by genetic modification to express bioactive molecule.Also can be when having bioactive molecule culture matrix.Can before being incorporated into substrate, CFU-GM in the process, or add bioactive molecule afterwards.The non-limiting ion of bioactive molecule comprises activin A, adrenomedullin, aFGF, ALK1, ALK5, ANF, angiogenin, angiopoietin-1, vascularization element-2, vascularization element-3, vascularization element-4, angiostatin, blood vessel attraxin, angiotensin-2, AtT20-ECGF, β cytokines, bFGF, B61, bFGF induced activity, cadherins, CAM-RF, cGMP analog, ChDI, CLAF, claudins, collagen, collagen receptor α ₁β ₁And α ₂β ₁Connect albumen, Cox-2, ECDGF (endotheliocyte derivative growth factor), ECG, ECI, EDM, EGF, EMAP, endoglin, Endothelin, endostatin, the endothelial cell growth inhibitor, the endotheliocyte vigor is kept the factor, endothelium differentiation sheath sugar g protein coupled receptor-1 (EDG1), ephrins, Epo, HGF, TNF-α, TGF-β, PD-ECGF, PDGF, IGF, IL8, growth hormone, fibrin fragment E, FGF-5, fibronectin, fibronectin receptor α ₅β ₁, factor X, HB-EGF, HBNF, HGF, HUAF, vascular cell amplification cardiac-derived inhibitor, IFN-γ, IL1, IGF-2 IFN-γ, integrin receptor (α subunit (α for example for example ₁, α ₂, α ₃, α ₄, α ₅, α ₆, α ₇, α ₈, α ₉, α _E, α _V, α _IIb, α _L, α _M, α _X) and β subunit (β for example ₁, β ₂, β ₃, β ₄, β ₅, β ₆, β ₇And β ₈) various combinations), K-FGF, LIF, the leiomyoma derivative growth factor, MCP-1, macrophage derived growth factor, the monocyte derived somatomedin, MD-ECI, MECIF, mmP2, mmP3, mmP9, the urokinase plasminogen activators, neuropil albumen (NRP1, NRP2), neurothelin, nitric oxide donors, nitric oxide synthetase (NOS), notch, closed albumen, banded closed albumen, oncostatin M, PDGF, PDGF-B, pdgf receptor, PDGFR-β, PD-ECGF, PAI-2, PD-ECGF, PF4, P1GF, PKR1, PKR2, PPAR-γ, PPAR-γ part, phosphodiesterase, prolactin antagonist, prostacyclin, protein S, smooth muscle cell derivative growth factor, the smooth muscle cell migration factor of deriving, sphingosine-1-phosphate-1 (S1P1), Syk, SLP76, tachykinin, TGF-β, Tie1, Tie2, the TGF-beta receptor, TIMP, TNF-α, TNF-β, transferrins, thrombospondin, urokinase, VEGF-A.VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF, VEGF ₁₆₄, VEGI, EG-VEGF, vegf receptor, PF4, prolactin antagonist 16kDa fragment, PGE1 and E2, steroid, heparin, 1-butyryl glycerol (only son's acid glyceride) and niacin amide.Other preferred embodiment in, substrate contains chemotherapeutant or immune modulatory molecules.These preparations and molecule are well known by persons skilled in the art.Preferably, substrate contains bFGF, VEGF or PDGF or their some combination (referring to embodiment 3; Embodiment 7).

Can regulate HSC-in the engineered tissue graft and the MSC angiogenesis of deriving according to the sustained release of somatomedin.As the result of the unusual high osmosis of endotheliocyte, engineering vascular tissue can be " leakage ".Can send the maturation that angiogenesis growth factor strengthens the HSC endotheliocyte by the microencapsulation of the Vascularised tissue graft of deriving at the HSC-that implants and MSC.

The bio-pharmaceutical that can join the present composition comprises that immunomodulator and other biological reply dressing agent.Biological response modifier generally includes and participates in modified biological and reply the biomolecule of immunne response for example or tissue or organ growth and reparation (peptide for example, the peptide segment, polysaccharide, lipid, antibody), it is with a kind of method that strengthens the certain desired treatment, for example lysis of bacterial cell, or the growth of tissue or organ specificity cell, perhaps vascularization.Bio-pharmaceutical also can be introduced directly in the matrix components.Those skilled in the art can understand or can determine easily that other can be used as the material of suitable abiotic medicine and bio-pharmaceutical.

Compositions of the present invention also can be modified to introduce diagnostic reagent, for example reagent of radip-opaque.The existence of these reagent makes the doctor can monitor the progress of the inner wound repair that takes place.These chemical compounds comprise barium sulfate, and the various organic compound that contains iodine.The example of latter's chemical compound comprises cholimil, iodipamide, iodoxamate meglumine, iopanoic acid and diatrizoate methylglucamine sodium.Other contrast agent that can use in compositions of the present invention can be determined easily by those skilled in the art, and can comprise the radiolabeled fatty acid of use or its analog.

The concentration of reagent will be according to the character of chemical compound in the compositions, physiological action and desired therapeutic or diagnosis effect and change.The treatment effective dose normally shows desired effects and does not have unsuitable toxic sufficient treatment reagent concentration.The diagnosis effective dose normally can the effective monitoring tissue grafts integration and make the concentration of the minimized diagnostic reagent of potential toxicity.In any case those skilled in the art determine the expectation concentration of specific compound in particular case easily.

Base composition can for example human serum albumin (HSA), hetastarch, dextran or its combination be strengthened or are strengthened by using fill-in.Also can by add non-degeneration nonionic detergent for example polysorbate80 strengthen the dissolubility of base composition.The debita spissitudo that is used for these chemical compounds of the present composition will be well known by persons skilled in the art, perhaps can not need too much experiment and determine easily.Base composition also can further be strengthened by using selectable stabilizing agent or solvent.It will be well known by persons skilled in the art suitably using these, perhaps can not need too much experiment and determine easily.

Implant

Engineered tissue of the present invention or organ compositions have significant clinical value, because compare with engineered tissue or organ by the similar stage that other means known in the art produced, they have the vascularization level of raising.This raising of vascularization has realized the regeneration of more effective tissue and organ, and this makes compositions of the present invention become the option that is different from other traditional treatments.

Usually evaluate determining that treatment needs by history and the physical examination consistent with related tissue or organ defective.Be accredited as the individuality that needs treatment and comprise having the tissue diagnosed or the individuality of organ defective.Preferred individuality is an animal, includes but not limited to mammal, reptile and birds, and more preferably horse, cattle, Canis familiaris L., cat, sheep, pig or chicken, optimum is chosen.

As an example, there is the individuality that needs to lack at least 5%, 10%, 25%, 50%, 75%, 90% or more particular cell types.As another example, there is the individuality that needs to have the damage of tissue or organ, and this method provides tissue or organ biological function to improve at least 5%, 10%, 25%, 50%, 75%, 90%, 100% or 200%, even reaches 300%, 400% or 500%.As another example, there is the individuality that needs can suffer from disease, disorder or disease, this method provide engineered tissue or organ construct, are enough to improve or stable disease, disorder or disease.For example, this individuality can suffer from disease, disorder or disease, and this causes the loss of cell, atrophy, dysfunction or death.The exemplary disease of being treated comprises nerve, neuroglia, or muscle degenerative disease, amyotrophy or nutritional disorder, the for example congenital heart failure of heart disease, hepatitis or liver cirrhosis, autoimmune disease, cancer, cause to remove the birth defect of tissue or organ, perhaps need for example angina pectoris of disease, disorder or disease of resection organization or organ, myocardial infarction and ischemic disease of limb, unexpected tissue disappearance or damage be fracture or wound for example.In further example, there is the individuality that needs can have the generation disease, the excessive risk of disorder or disease, the present invention can postpone or prevent its generation.

Tissue or organ can be selected from bladder, brain, nervous tissue, neuroglia, esophagus, fallopian tube, heart, pancreas, intestinal, gallbladder, kidney, liver, lung, ovary, prostate, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, urogenital tract, ureter, urethra, uterus, breast, skeletal muscle, skin, bone and cartilage.Blood vessel CFU-GM and/or organize the CFU-GM can be from the individual individuals with same of transplanting with the engineered tissue compositions.Interchangeable, CFU-GM can be from identical species, perhaps even not same species.

Implant the technical ability that engineered tissue or organ construct belong to this area.Substrate and cell assemblage can be implanted in individual tissue or the organ to become its funtion part wholly or in part.Exchange in conjunction with the host or with the host by cell monolayer when preferably, implant begins.Through after a while, the cell of being introduced can increase and move out polymeric matrix and reach surrounding tissue.After implantation, the cell around the engineering blood vessel tissue compositions can enter by cell migration.Cell around the engineered tissue can be attracted by bioactive materials, comprise biological response modifier, polysaccharide for example, protein, peptide, gene, antigen and antibody that can the selectivity doped matrix are to provide needed selectivity, for example, cell receptor is limited to substrate, or irritation cell migration enters substrate, perhaps the two.Usually, substrate is porous, and it has interconnective microchannel and/or major path, and it can realize the cell migration that amplifies by biological and physical chemistry gradient.For example, the cell that centers on the substrate of implanting can be attracted by bioactive materials, comprises VEGF, fibroblast growth factor, transforming growth factor-beta, endothelial cell growth factor (ECGF), one or more of palatelet-selectin and ICAIU.Those skilled in the art can accept and know how to use other to be suitable for the bioactive materials of cytotaxis on the substrate.

Biomolecule can be introduced substrate, so that biomolecule embeds in it.Interchangeable, can use chemical modification method with surperficial covalently bound biomolecule in substrate.By using for example aldehyde compound of coupling agent as known in the art, imidodicarbonic diamide etc., the surface functional group of matrix components can form covalent bond with functional group's coupling of biomolecule.In addition, can use the sept molecule to be used for interrupting the active group of collagen surface active groups and biomolecule, so that these molecules on the stromal surface are more pliable and tougher.Biomolecule is attached to substrate inside or other outside similar approach will be well known by persons skilled in the art.

Method of the present invention, compositions and equipment can comprise simultaneously or use in enzyme, ion, somatomedin and the biological reagent one or more successively for example thrombin and calcium or its combination are handled.Method of the present invention, compositions and equipment can comprise abiotic medicine of use and/or bio-pharmaceutical while or handle successively.

Screening

Another aspect of the present invention provides a kind of method of screening the molecule of modulating vascular formation.This method comprises the following steps: and will organize CFU-GM and blood vessel CFU-GM to be incorporated into host material; The culture matrix material is to form engineered tissue; Host material or engineered tissue are contacted with candidate molecules; Measure the vascularization of engineered tissue; And determine with respect to the contrast that does not contact candidate molecules whether candidate molecules regulates and control substrate/the organize formation of medium vessels.Randomly, this screening technique also can comprise host material or engineered tissue are incorporated in the individuality, and induce endogenous tissue CFU-GM and/or blood vessel CFU-GM to move in the construct of being implanted.

Preferably, candidate molecules is the part of test mixing thing, for example cell pyrolysis liquid, the lysate that comes self-organizing or library.Compare with the individuality that does not contact accordingly this molecule, the molecule that modulating vascular forms can improve or reduce vascularization (angiogenesis for example in culture, substrate, tissue or organ, vascularization, the formation of immature blood vessel network, blood vessel remodeling, the blood vessel stabilisation, mature blood vessel, blood vessel differentiation, or set up the functional type blood vessel network) at least 5%, 10%, 20%, 25%, 30%, 40% or 50%, 60%, 70%, 80%, 90%, perhaps even nearly 100%, 150% or 200%.

Through describing the present invention in detail, be apparent that some are modified, change and embodiment of equal value is possible, and do not leave scope of the present invention defined in the claim in the back.And, need all embodiment that understand herein all to provide as non-limiting example.

The list of references of being quoted

By with reference to all publications of being quoted among the application, patent, patent application and other list of references integral body are incorporated this paper into, reach as specifically and individually describing each piece here by incorporate the publication of this paper into reference to integral body, patent, the Chengdu that patent application is the same with other lists of references.This paper incorporated by reference document should not be considered as admits that these are prior aries of the present invention.

Embodiment

Non-limiting example below providing is to further specify the present invention.On behalf of the present inventor, the disclosed inventor's technology among the embodiment in the back of it will be appreciated by those skilled in the art that have been found that in the approach of implementing to give full play to when of the present invention function, therefore has been considered to constitute the example of its Implementation Modes.Yet those skilled in the art can understand and can carry out many changes to the disclosed specific embodiment, and still can obtain identical or similar result according to disclosure of the present invention, and this does not leave the spirit and scope of the invention.Should be appreciated that described in an embodiment any method can actually be carried out or not carry out, perhaps any compositions of describing in an embodiment can be actual formation or not formation, and this employed verb time sequence is irrelevant.

Embodiment 1: the endotheliocyte with MSC osteoblast co-inoculation on the space has produced capillary structure in the engineering bone construct in vivo

Prepare people's bone marrow sample (AllCells, Berkeley, CA), according to the method for setting up before (people such as Shi, 1998; People such as Alhadlaq, 2004; People such as Yourek, 2004; People such as Marion, 2005; People such as Moioli, 2006; Troken and Mao, 2006) separating mesenchymal stem cell (MSC) and hematopoietic stem cell (HSC).Figure 1A has described the initial bone marrow content of paving plate, its show dense known be allogenic cell mass.(referring to Alhadlaq and Mao, 2004; Marion and Mao, 2006).

Mescenchymal stem cell can be divided into osteoblast.Two kinds of different cell lineage human mesenchymal stem cells (MSC) and Human umbilical vein endothelial cells (HUVEC) are used in the body in the vascularization bone engineering.

According to recited above, from people's bone marrow sample, separate MSC (referring to for example Figure 1B) (Alhadlaq and Mao, 2003; People such as Alhadlaq, 2004; People such as Yourek, 2004; Alhadlaq and Mao, 2005; People such as Moioli, 2006; Marion and Mao, 2006; Troken and Mao, 2006).The hMSCA of amplification cultivation is divided into osteoblast (people such as Marion, 2005; People such as Moioli, 2006).It is male to alkali phosphatase (referring to for example Fig. 1 C) and Feng Kusa dyeing (referring to for example Fig. 1 D) that hMSC is derivatized to osteocyte (hMSC-Ob).

HMSC is derivatized to osteocyte (5 * 10 ⁶Individual cell/mL) is seeded in porous surface (the β TCP of the bata-tricalcium phosphate dish in the slight decompression; Average pore size: 300 μ m) (referring to for example, Fig. 2 A, rose pink zone).

Endotheliocyte and MSC osteoblast co-inoculation are in vivo in the engineering bone construct.Human umbilical vein endothelial cells (HUVEC) is amplified cultivation, then with 5 * 10 ⁶The density of individual cell/mL is encapsulated under 4 ℃ of slight decompressions in the liquid phase of Matrigel (referring to for example Fig. 2 A, red point) equally.Matrigel is the counterdie macromolecule hydrogel, and it has been widely used in that endotheliocyte adheres to and angiogenesis research (people such as Abilez, 2006; People such as Baker, 2006; People such as Bruno, 2006; People such as Mondrinos, 2006; People such as Rajashekhar, 2006).

HUVEC-Matrigel construct (referring to for example Fig. 2 A, red point) is advanced by inoculation hMSC to derive in the hole of osteoblastic β TCP dish by infusion.Subsequently, make the Matrigel polymerization by hatching at 37 ℃.Infusion has the compound construct of HUVEC, and the β TCP construct (referring to for example Fig. 2 A) that will inoculate hMSC-Ob is implanted to 4 weeks of back of strict combination immunodeficiency (SCID) mice.The contrast construct comprises β TCP dish and the acellular β TCP dish of inoculating hMSC-Ob.

After the results body was implanted into, the β TCP construct of the infusion HUVEC of institute of recovery, inoculation hMSC-Ob showed the timbering material (referring to for example Fig. 2 B) of β TCP in mineralising area and the Feng Kusa stained.By hematoxylin and eosin dyeing (referring to for example Fig. 2 C), in the mineralising tuberosity, find the formed blood vessel class of endothelium class cell inner chamber (referring to for example, the PV among Fig. 2 C).By the Feng Kusa stained of high-amplification-factor more, a large amount of zones of β TCP construct are by mineralising (referring to for example Fig. 2 D).If HUVEC is seeded among the Matrigel equably, then the reconstruction that the back participates in HUVEC is obviously implanted in the formation (referring to for example Fig. 2 C) of the inner chamber class formation lined up of endothelium class cell in vivo.

These digital proofs can be mediated capillary structure by the people MSC osteoblast and the human endothelial cell of co-inoculation in biocompatible materials different spaces zone in mineralized tissue.Therefore, many cell lineages can be optimised in engineering vascularization bone, and for example HSC, MSC and/or their pedigree derivant comprise that derive endotheliocyte and MSC of HSC is derivatized to osteocyte.

Embodiment 2: the bone marrow derived hematopoietic stem cell becomes endothelium class cell in vitro differentiation.

For clinical practice, can from bone marrow, separate HSC, preferably by bottom line invasive approach together with MSC.Have been found that HSC has experienced slow amplification (people such as Shih, 2000; People such as Li, 2004).FGF-2 has been proved to be and can have quickened HSC amplification rate (Wilson and Trump, 2006; People such as Yeoh, 2006).The inventor's experience is that HSC is really to increase than MSC and the slow speed of HUVEC.Alternative, HSC can be divided into endotheliocyte, the HSC that then the increases endotheliocyte of deriving.

Preparation bone marrow sample (ditto) is used to separate HSC.CD34 separate with magnetic bead be used to separate NAC (EasySep.AllCells, Berkeley, CA).The isolating CD34 positive cell of institute (CD34+) is considered to HSC.In applying the flat board of fibronectin, HSC is shown as round cell (referring to for example Fig. 3 A), its shape with in the 2D cultivation, present fusiform MSC obviously different (referring to for example Figure 1B).After HSC is transferred to new culture plate, the endothelial cell differentiation fill-in is joined DMEM, it contains VEGF (10ng/mL), bFGF (1ng/mL) and IGF-1 (2ng/mL) (people 1998 such as Shi; People such as Shih, 2000; People such as Li, 2004).

HSC begins to form colony (referring to for example Fig. 3 B) in about 2 weeks.Further, under the stimulation of endothelium differentiation fill-in, HSC is divided into the cell that does not connect, and it forms the tubular structure (referring to for example Fig. 3 C) of interconnected cell.Locating in the cell as Ac-LDL fluorescence is proved, the HSC derived cell is male to acetylation low density lipoprotein, LDL (Ac-LDL), and wherein Ac-LDL is a kind of typical endothelial cell marker thing (referring to for example Fig. 3 D).As what antibody staining proved, the HSC endotheliocyte of deriving is also expressed von willebrand's factor (vWF), a kind of label of natural endotheliocyte (referring to for example Fig. 3 E).HSC derives the comparison of endotheliocyte (HSC-EC) expression according to the remarkable high vWF (referring to for example Fig. 3 F, left post) of fibroblast (FB) (referring to for example Fig. 3 F, right post).

In sum, these digital proofs are proved as natural endothelial cell morphology and label, can be divided into endotheliocyte class cell from the isolating HSC of people's bone marrow.The deutero-endotheliocyte of these HSC forms the iuntercellular tubulose and connects.

Therefore, can be derivatized to osteocyte by fusion HSC and MSC and/or the deutero-endotheliocyte of HSC and MSC and produce engineering vascularization bone.This has simulated in growth course how to invade the formation nature bone by blood vessel.The osteogenesis in key district, the middle part of long bone has blood vessel, and this has proved hematopoietic stem cell and the synergism of mescenchymal stem cell in natural (vascularization) angiogenesis admirably.

Embodiment 3: somatomedin is induced the angiogenesis in the macromolecule hydrogel in vivo.

This paper has proved that HSC and MSC can be divided into final cell pedigree for example endotheliocyte and osteoblast, and it has constituted the construct assembly of some blood vessel and bone.Proved that also capillary structure can be built upon in the bone support in vivo.Yet existing literature has shown that the engineering blood vessel may be owing to unusual high endotheliocyte permeability but (people such as Richardson, 2001 of leaking; Valeski and Baldwin, 2003).In order to determine the influence of bFGF to the angiogenesis of host derivation, angiogenesis factor bFGF is delivered to intensive macromolecule hydrogel, in poly-(ethylene glycol) diacrylate (PEGDA), in the former research known its be impermeable (Alhadlaq and Mao, 2003 to the host derivation vascular cell in vivo; People such as Alhadlaq, 2004; Alhadlaq and Mao, 2005; Stosich and Mao, 2006).

When tissue engineered bone is enlarged greatly to repair towards clinical practice, during the bone defective of serious size, the vascularization meeting of suboptimum is problematic especially.Below shown digital proof physics major path and the bioactie agent that is encapsulated in the macromolecule hydrogel induce the deutero-angiogenesis of host.

Four kinds of structures (referring to for example Fig. 4) (people such as Stosich, 2006) in the PEG hydrogel, have been designed.Group 1 is made of separately the PEG hydrogel.Size with 6 * 4mm (diameter * thickness) forms PEG cylinder (referring to for example Fig. 4 A).Group 2 is made of separately major path.In photopolymerization PEG cylinder, form 3 major paths (diameter is 1mm) (referring to for example Fig. 4 B) altogether.Group 3 is made of separately bFGF.In the liquid phase of PEG hydrogel, load 10 μ g/mLbFGF altogether, then carry out photopolymerization.In this group, do not form major path (referring to for example Fig. 4 C).Group 4 is made of bFGF and major path.In the liquid phase of PEG hydrogel, load 10 μ g/mLbFGF altogether, then carry out photopolymerization and form 3 major paths (diameter is 1mm) (referring to for example Fig. 4 D).In any four groups, all do not send foreign cell.All PEG cylinders all have 6 identical * 4mm (diameter * thickness) size, and are implanted into 4 week of back of SCID mice by subcutaneous body.

Implant immunodeficient mouse in vivo after interior 4 weeks, the sample analysis that passes through to be extracted has been noticed following.The PEG hydrogel that bFGF or major path are not set demonstrates the macroscopical evidence (referring to for example Fig. 5 A) that does not have the blood vessel infiltration.The PEG hydrogel that has 3 physics major paths on the contrary demonstrates 3 red points (referring to for example Fig. 5 B) when gathering in the crops in vivo.Following histology and immunohistochemistry evidence hint that these contain the vascular tissue of host derivation.Load bFGF but do not have on the PEG hydrogel integral color of major path darker (referring to for example Fig. 5 C).The random areas of following histology and the infiltration of immunohistochemistry evidence hint host derivation vascular tissue.PEG hydrogel with major path and load bFGF is not only darker on the integral color, and also demonstrates 3 red points (referring to for example Fig. 5 D) results the time in vivo.Below histology and immunohistochemistry evidence proof histiocyte only penetrate in the inner chamber of major path, and not in other positions of PEG hydrogel.

Histology and immunohistochemistry find that people (2006) such as () Stosich is as follows.The PEG hydrogel (group 1) that does not have bFGF or major path shows the sign (referring to for example Fig. 6 A) that does not have host cell infiltration or any angiogenesis, this and before data consistent (Alhadlaq and Mao, 2003; People such as Alhadlaq, 2004; Alhadlaq and Mao, 2005; Stosich and Mao, 2005).Have major path but do not have the PEG hydrogel (top group 2) of bFGF to prove that host cell only penetrates in the major path, and not in other parts (referring to for example Fig. 6 B) of PEG.On the contrary, load bFGF and do not have the PEG hydrogel (group 3) of major path to demonstrate obviously at random zone (referring to for example Fig. 6 C) of host cell infiltration.Load bFGF and the PEG hydrogel (top group 4) with major path prove that host cell only penetrates in the major path, and not in other parts (referring to for example Fig. 6 D) of PEG.

These results show following.Have the two PEG hydrogel of major path and bFGF and have 0.47 ± 0.18mm ²The inside increment of host tissue, this does not have 0.13 ± 0.05mm of the PEG hydrogel of bFGF significantly than having major path ²High (meansigma methods ± S.D.; P＜0.01; 8 every group of N=) (referring to for example Fig. 7).Therefore physics and the biological activity modular design in the PEG hydrogel can promote host tissue inwardly to grow.

More the analysis of high power image shows that blood vessel penetrates into the PEG hydrogel, but can resist host tissue inwardly grow (referring to for example Fig. 8).

In the host tissue that has or do not have bFGF, take place inwardly to grow (referring to for example Fig. 8 A, 9B and Fig. 8 E, 9F).Yet as shown in FIG. 7, the amount of the host tissue that in the major path of the PEG of load bFGF hydrogel, is permeated (referring to for example Fig. 8 E, 9F) significantly greater than do not have bFGF have major path PEG hydrogel (referring to for example. Fig. 8 A, 9B).Load bFGF but do not have the PEG hydrogel of major path to demonstrate rare tissue ingrowth (referring to for example Fig. 8 C, 9D).Capillary structure is in the capillary structure that is arranged in by endothelium class cell and is centered on by fibroblast class cell, and the cell that contains similar erythrocyte is (referring to for example Fig. 8 E, 9F).

Use the immunolocalization of anti-vascular endothelial growth factor (VEGF) antibody staining to show that ingrown host tissue is a vascular tissue.The strong anti-VEGF of appearance dyes (referring to for example Fig. 9 B, 10D) in the host tissue that is permeated in the major path of bFGF having or not having.VEGF antibody is tagged tissue capsule (referring to for example Fig. 9 A) and be penetrated into host tissue in the PEG hydrogel that has bFGF but do not have major path also.(referring to for example Fig. 9 C).

These data acknowledgement capillary structures (as for example shown in Fig. 8) are by bFGF in the PEG hydrogel and/or the inductive host derivation angiogenesis of major path.In not having the PEG hydrogel of bFGF or major path, there is not angiogenesis (referring to for example Fig. 9 A).As the middle lipoblast of formerly working, become cartilage and osteoblastic survival to be proved, the hole of PEG hydrogel may be enough greatly to allow the diffusion of somatomedin and nutrient (people such as Burdick, 2003; People such as Kim, 2003; People such as Alhadlaq, 2004; Alhadlaq and Mao, 2005; People such as Moioli, 2006; Stosich and Mao, 2006).Yet the size in the aperture of PEG hydrogel is not enough to allow host cell inwardly to grow, unless introduce for example bFGF of passage and somatomedin.

Therefore, the inside growth of host tissue can be used for guiding angiogenesis and host cell along predetermined route invasion in major path.In addition, the amplification of bFGF or other angiogenesis factors helps further to quicken inwardly growth.The maturation that these are found to support the adjusting of host derivation angiogenesis and strengthen engineering blood vessel in the bone construct.

Embodiment 4: the cell inoculation density in the organizational project (engineering)

Very actual problem is that how many cells are introduced in the support (Moioli and Mao, 2006) in engineering (engineering) biological structure.When mescenchymal stem cell produced into the osteoblast of osteoprogenitor cells and final stage in growth, fissional density dependency suppressed the factor that (being known as contact inhibition) is cell survival before people such as (, 2002) Alberts.The too much cell of inoculation may cause the part can utilize the shortage of mitogen, somatomedin and survival factors, the unnecessary waste (Moioii and Mao, 2006) that may cause apoptosis and cause the cell in vitro proliferation time in the engineered tissue support.On the other hand, inoculation cell very little in the engineered tissue support, it is poor as a result to cause regenerating.Therefore, need to determine the optimum density of HSC, MSC and their pedigree derivants, so that the regeneration result of engineering vascularization bone maximization (referring to for example Figure 10).

Here reported that MSC, MSC are derivatized to the derive influence of various initiator cell density of chondrocyte of osteocyte and MSC.Separation of human MSC from each of the multiple medullary cell sample of a plurality of healthy donors increases in monolayer culture, and as above and according to existing method is divided into chondroblast and osteoblast (people such as Alhadlaq, 2004 respectively; People such as Marion, 2005; People such as Yourek, 2005; People such as Moioli, 2006) (referring to for example. Figure 10).To every kind of cell lineage hMSC, hMSC-Ob and hMSC-Cy adopt four kinds of cell densities: 0 * 10 ⁶Individual cell/mL, 5 * 10 ⁶Individual cell/mL, 40 * 10 ⁶Individual cell/mL and 80 * 10 ⁶Individual cell/mL.Studied intermediary cell inoculation density 20 * 10 in the past ⁶Individual cell/mL (Alhadlaq and Mao, 2005).0 * 10 ⁶Individual cell/mL=does not have the construct of cell.The cell suspending liquid of every kind of cell density and pedigree is encapsulated in the aqueous phase of PEG hydrogel, then carries out photopolymerization, and continuous culture 3D PEG 4 weeks of construct (referring to for example Figure 10).

Replenish DMEM or becoming cartilage to replenish among the DMEM at DMEM, skeletonization respectively, utilizing the frequent culture medium continuous culture 3D PEG hydrogel construct of changing after 4 weeks, carrying out tissue staining and biochemical test.The skeletonization culture medium contains 100nM dexamethasone, 50 μ g/ml ascorbic acid and 100mM β-phosphoglycerol, forms the cartilage supplementing culture medium and contains 10ng/mlTGF β 3 (details is as follows).

The result be presented at corresponding D MEM, skeletonization replenish DMEM or become cartilage to replenish hatched for 4 weeks in the DMEM culture medium after, in the PEG hydrogel, keep initial cell inoculation density (referring to for example Figure 11) (referring to for example, Troken and Mao, 2006).Figure 11 has described H﹠amp; The painted exemplary results of E, and the hMSC (row of going up), the hMSC that are presented in the PEG hydrogel encapsulation and carry out the various density that 4 week 3D constructs cultivate are derivatized to the derive histological observation of chondrocyte (arranging down) of osteocyte (middle row) and hMSC.Usually, the terminal point cell density has been followed similar initiator cell inoculum density pattern in the PEG hydrogel support: 5M cell/ml, 40M cell/ml and 80M cell/ml (5,000,000 cells of the every ml cells suspension of 5M/ml=).

After in the PEG hydrogel, hatching for 4 weeks, the hMSC chondrocyte (hMSC-Cy) of deriving has not only kept their one-tenth cartilage phenotype, but also kept their corresponding initiator cell inoculum density (referring to for example. Figure 12, safranin O dyeing) (referring to for example, Troken and Mao, 2006).Safranin O is the dye of positive ion, and it is in conjunction with the relevant mucopolysaccharide of cartilage for example keratin sulfate and chondroitin sulfate, and be widely used in labelling natural joint and the dull and stereotyped cartilage of growth (referring to for example, people such as Mao, 1998; Wang and Mao, 2002; Sundaramurthy and Mao, 2006).Yet, in the PEG hydrogel, hatched for 4 weeks after, although hMSC has kept their initial inoculum density, they are negative (referring to for example Figure 12) to safranin O dyeing.

After in the PEG hydrogel, hatching for 4 weeks, hMSC is derivatized to osteocyte (hMSC-Ob) and has not only kept their one-tenth bone phenotype, but also kept their corresponding initiator cell inoculum density (referring to for example Figure 13, Feng Kusa dyeing) (Troken and Mao, 2006).Feng Kusa dyeing be normally used for mineralising in natural skeletonization of labelling and the organizational project skeletonization form (referring to for example, Alhadlaq and Mao, 2003; People such as Alhadlaq, 2004; People such as Marion, 2005; People such as Moioli, 2006).Yet, in the PEG hydrogel, hatched for 4 weeks after, although hMSC has kept their initial inoculum density, they are negative (referring to for example Figure 13) to Feng Kusa dyeing.

After the PEG hydrogel of implanting encapsulation equal densities hMSC, hMSC-Ob and hMSC-Cy is in the nude mice, the hMSC that the cell inoculation density that data show improves in the body can cause improving is derivatized to the derive amount (referring to for example Figure 14) of substrate that chondrocyte forms of osteocyte and hMSC, this vitro data that is provided above having continued (referring to for example, Figure 11-14).

This cell density experimental verification before by two kinds of cell densities 5 * 10 relatively ⁶Individual cell/mL and 20 * 10 ⁶Individual cell/mL (people such as Alhadlaq, 2004; Alhadlaq and Mao, 2005) discovery in the regeneration result of high cell inoculation density for example 20 * 10 ⁶Individual cell/mL is better than 5 * 10 ⁶The inoculum density of individual cell/mL.Yet too high cell inoculation density can cause organizes for example nutrient shortage, the unusual and unnecessary waste of cell in vitro proliferation time (Moioli and Mao, 2006) of iuntercellular contact.Usually the preferred the shortest proliferation time that exsomatizes.

These cell densities experimental results show that the optimization of cell inoculation density packaged in tissue engineering bracket can make regeneration result maximization (referring to people such as Alhadlaq, 2004; Alhadlaq and Mao, 2005; Troken and Mao, 2006).

Optimal proportion between embodiment 5:HSC and the MSC

HSC of following experimentation vascularization bone engineering the best and the ratio between the MSC.

Table 2

Usage factor method for designing research HSC and MSC are to the Relative Contribution of vascularization bone engineering in 8X8X2 design: cell proportion (8) * sample size (8) * body is implanted into the time (2).The total number of cell in external support (HSC and MSC combination) is held constant at 8 * 10 ⁶Individual cell/mL, and therefore the relative scale of HSC and MSC can determine HSC and the MSC Relative Contribution to engineering vascularization bone between 1:1～1:15.

According to research recited above and the method set up before, separation of human HSC and MSC (Alhadlaq and Mao, 2003 from each of multiple bone marrow sample; People such as Alhadlaq, 2004; People such as Yourek, 2004; Alhadlaq and Mao, 2005; Moioli and Mao, 2006; People such as Moioli, 2006; Marion and Mao, 2006; Troken and Mao, 2006; People such as Stosich, 2006).HSC and MSC from single donor are used in every kind of construct to eliminate any possible immunologic rejection problem.As above-mentioned research, HSC by in the even inoculation in Matrigel, and be infused in the hole of the β TCP that is inoculated MSC in advance.The cytoskeleton construct is implanted to the back that can not repel the nude mice of people's cell.The principle that body is implanted into 8 weeks and 16 weeks is according to previous experience, if take place, then is desirably in this time limit and takes place people such as (, 2006) Stosich.

The sample implanted of results, and carry out the listed analysis of following table 3.

The product test and the successful standard of table 3. engineering vascularization bone.The details method of these technology is discussed in the back

H﹠amp; E: hematoxylin and eosin, global tissue are learned color and are used to distinguish multiple tissue; Masson trichrome stain: the histological stain of blood vessel; OCN: osteocalcin, osteoblastic attachment proteins, the label in late period of Osteoblast Differentiation; OPN: osteopontin, osteoblastic attachment proteins, the label in late period of Osteoblast Differentiation; VWF: von willebrand's factor, the surface glycoprotein of on endotheliocyte, finding, the label in late period of endothelial cell differentiation; VEGFR: vascular endothelial growth factor receptor, the label in late period morning of endothelial cell differentiation; KDR/VEGFR-2/Flk-1: vascular endothelial growth factor receptor 2, the label in late period morning of endothelial cell differentiation.

According to following described detailed method numeral X-ray and μ CT engineering vascularization bone is carried out quantitatively.Compression and shear force test the carrying out mechanical analysis of engineering vascularization bone that use utilizes the minute-pressure of atomic force microscope (AFM) and uses the traditional mechanical test.The microcosmic mechanical performance of engineering vascularization bone is interested, and is studied by AFM easily, but can not obtain by the macro-size mechanical test that utilizes Instron or MTS.Yet, reduced overall and shear force performance that MTS can testing engineering vascularization bone, this can not test by AFM.Therefore, AFM and MTS are the complementary mechanical test approach of engineering vascularization bone.The data of all numerals all are carried out statistical analysis.For the data normal distribution, use the variance analysis (ANOVA) that utilizes the Bonferroni check.If DATA DISTRIBUTION is asymmetric, then use for example Kruskal-Wallis variance analysis of non parametric tests.Statistical significance is 0.05 alpha levels.

Autogenous cell and homogeneous variant cell all are used in the organizational project.This paper provides the model (implanting the people's cell in the nude mice) of autogenous cell in the organizational project.Nude mice is as anthropomorphic dummy " couveuse ".Compare with homogeneous variant cell, autogenous cell has many important advantages, for example lacks immunologic rejection and pathogen propagation.Homogeneous variant cell can be made into to be suitable for receptor easily, has therefore saved the required time of the cell manipulation relevant with autogenous cell.Yet, may need the administration immunosuppressive drug, and may make the organizational project complexity as a result of vascularization bone.The selection of bone marrow stem cell be to small part based on observe as in the described research in the above prove that bone marrow derived MSC and HSC have fully been identified, and had the ability of setting up the vascularization bone.The adipose-derived stem cell is in the news recently, and the succedaneum of bone marrow derived cell can be provided.

The optimum cell density of embodiment 6:HSC, MSC and their pedigree derivant makes the maximum production of engineering vascularization bone.

Although HSC and MSC be collaborative performance function in the vascularization bone development, also have some other cell line also to participate in the blood vessel osteogenesis and comprise endotheliocyte and osteoblast.Osteoblast is one of cell of the deutero-final stage of a kind of MSC.Therefore, whether need study vascularization osteogenesis engineering can be derivatized to osteocyte and MSC and HSC by fusion HSC and MSC and derive endotheliocyte and maximize.Whether endotheliocyte is derived from MSC, HSC or other CFU-GM, is not also understood (Yin and Li, 2006) fully.Endothelium class cell is from HSC differentiation, therefore provides the living cells source to be used to study the HSC endotheliocyte of deriving and participated in engineering vascularization bone.

Below the cell inoculation density of Research on experiment design in vascularization bone engineering, be not only HSC and MSC, also have their pedigree derivant to comprise that derive endotheliocyte and MSC of HSC is derivatized to osteocyte.

Table 4: experimental design, experiment 1-HSC and MSC are derivatized to osteocyte.Usage factor method for designing research HSC and MSC are derivatized to the Relative Contribution in the osteocyte vascularization bone engineering in 8 * 8 * 2 designs.Cell proportion (8) * sample size (8) * body is implanted into the time (2).

Table 5. experimental design, experiment 2-MSC and the HSC endotheliocyte of deriving.Usage factor method for designing research MSC and the HSC Relative Contribution in the endotheliocyte vascularization bone engineering of deriving in 8 * 8 * 2 designs.Cell proportion (8) * sample size (8) * body is implanted into the time (2).

According to research recited above and the method set up before, separation of human HSC and MSC (Alhadlaq and Mao, 2003 from each of multiple bone marrow sample; People such as Alhadlaq, 2004; People such as Yourek, 2004; Alhadlaq and Mao, 2005; Moioli and Mao, 2006; Marion and Mao, 2006; Troken and Mao, 2006; People such as Stosich, 2006).HSC and MSC from single donor are used in every kind of cell inoculation construct to eliminate possible immunologic rejection problem.For experiment 1, according to the approach of setting up before, MSC is divided into osteoblast class cell (Alhadlaq and Mao, 2003; People such as Alhadlaq, 2004; People such as Yourek, 2004; Alhadlaq and Mao, 2005; Moioli and Mao, 2006; Troken and Mao, 2006; Marion and Mao, 2006).For experiment 2, according to the approach in the described research in the above, HSC is divided into endotheliocyte class cell.Research as described above, the HSC endotheliocyte of deriving evenly is seeded among the Matrigel, and is infused into and is inoculated MSC in advance and derive in the hole of osteoblastic β TCP.For experiment 2, before endotheliocyte that HSC is derived is seeded in Matrigel, at first MSC is seeded in the hole of β TCP.For experiment 1 and 2, the cytoskeleton construct is implanted to the nude mice that can not repel people's cell.The principle that body is implanted into 8 weeks and 16 weeks is according to previous experience, if take place, then is desirably in this time limit and takes place people such as (, 2006) Stosich.

Product test and data analysis and statistics are as mentioned above.

Inoculate derive endotheliocyte and MSC is derivatized to osteocyte or chondrocyte also can take place of HSC simultaneously.

Embodiment 7: angiogenesis growth factor promotes the derive maturation of vascularization bone medium vessels at HSC-and MSC.

The engineering vascular system must correctly be brought into play function and for example provide suitable nutrient supply in the new osseous tissue that forms, oxygenation, and gas exchange and cell are supplied with.Vascularization comprises a series of cascade of events, and it comprises that endotheliocyte activates, moves and propagation.The engineering blood vessel may since unusually high permeability leak (people such as Richardson, 2001; Valeski and Baldwin, 2003).Known many angiogenesis growth factors are regulated formation (Thurston, 2002 of blood vessel in natural growth; People such as Ehrbar, 2003; Valeski and Baldwin, 2003; Ferrara, 2005).Several days VEGF of the beginning that generates at the bone medium vessels highly express (people such as Nissen, 1996; People such as Hu, 2003; Bohnsack and Hirschi, 2004; Ferrara, 2005).After the VEGF effect, PDGF influences vascular system, and strengthens maturation (Darland and D ' Amore, 1999 of vascular endothelial cell; People such as Richardson, 2001; Bohnsack and Hirschi, 2004).The reason of " leakage " blood vessel is because a small amount of relevant for example peripheral cell of parietal cell and smooth muscle cell in the organizational project.PDGF has shown recruitment (Darland and D ' Amore, 1999 of inducing parietal cell; People such as Yancopoulos, 2000; Valeski and Baldwin, 2003; Ferrara, 2005).Therefore, send PDGF also at making engineering neovasculature maturation by recruiting parietal cell.

In order to identify that VEGF and PDGF are inducing from the optimal dose in the maturation of the engineering blood vessel of HSC and HSC derived cell, explore the high and low dosage than known physiological dose.In the recruitment of angiogenesis cell and propagation, rapid release VEGF is (people such as Nissen, 1996 of expectation; People such as Hu, 2003; Ferrara, 2005).Therefore, VEGF is inhaled into beginning several hrs or several days rapid release to implant in vivo in the β TCP dish.PDGF acts on after the VEGF, and not only promotes the maturation of endotheliocyte, and as chemotactic factor (Darland and D ' Amore, 1999 of parietal cell; People such as Yancopoulos, 2000; Valeski and Baldwin, 2003; Ferrara, 2005).Therefore, PDGF is encapsulated in and is used in the microsphere continue discharging and does not have initial pulse phase people such as (, 2006) Moioli, so that can progressively continue to discharge PDGF after the VEGF performance function of rapid release more.According to the experience in the top described research, encapsulation PDGF microsphere will further delay its rate of release in Matrigel.

Table 6. is used for the sophisticated experimental design of enhancement engineering bone neovasculature.HSC-EC: the hematopoietic stem cell endotheliocyte of deriving; MSC-Ob: mescenchymal stem cell is derivatized to osteocyte.Usage factor method for designing result of study in 8 * 5 * 2 designs: sample size (8) * somatomedin dosage (5) * body is implanted into the time (2).

VEGF is inhaled among the Matrigel, then is infused in the hole of β TCP, carries out rapid release.Two emulsifying technologies of describing in this article by ins and outs and according to existing method, PDGF is encapsulated in (people such as Moioli, 2006) in the PLGA microsphere.There is not the initial pulse phase to discharge PDGF with speed slowly.Before growth factor-loaded, the program of cell inoculation is identical with embodiment 1.

Product test and data analysis and statistics are as mentioned above.

From above-mentioned and existing document, obtain VEGF and PDGF dosage (referring to for example, Darland and D ' Amore, 1999; People such as Yancopoulos, 2000; People such as Richardson., 2001; Valeski and Baldwin, 2003; Ferrara, 2005).Interchangeable, can use bFGF to replace VEGF, also be according to previous experience people such as (, 2006) Stosich.Increase multiple somatomedin and carry out cell and send and formed complicated system, how to take place although this is natural angiogenesis and skeletonization.The replacement that VEGF is sucked Matigel is lyophilizing β TCP.PLGA notified the degraded process in produce acidic by-products.Yet owing to have only a little P LGA to be used to make microsphere, so the acidic by-products problem is not important, and reached minimum people such as (, 2006) Moioli in the work formerly.Proved that as existing document PDGF is considered to recruit vascular smooth muscle cell (Darland and D ' Amore, 1999; People such as Yancopoulos, 2000; Valeski and Baldwin, 2003; Ferrara, 2005).Consider the economy of final clinical treatment, adopt minimum effective dose usually.By introducing HSC and MSC to engineering vascularization bone, the height when amount that might required angiogenesis growth factor is not so good as not introduce HSC and MSC (and/or pedigree derivant).In logic, HSC and MSC and/or their pedigree derivant also may mediate necessary angiogenesis growth factor.

The braincap defective of repairing serious size is effectively sent in the optimization of embodiment 8:HSC, MSC and/or angiogenesis growth factor.

Experiment described above provides the approach of optimizing based on cell and/or somatomedin to be used to use the engineering vascularization bone of dystopy skeletonization approach.The cranium defective has been represented the important clinical demand, the coordination site based on the approach of cell and/or somatomedin of having represented also that test optimizes in engineering vascularization bone.

The braincap defective that this experiment provides optimized conditions that whether coordination bone defective environment determine by the method for listing above with test more effectively to repair serious size than any independent composition and/or traditional bone tissue engineer approach.The braincap defective represented with described experiment in the above in the different experimental model in homotopic transplantation site that utilized.

Table 7. experimental design is used to utilize the braincap defective of the serious size of approach reparation of the engineering vascularization bone of optimization; MSC-Ob: mescenchymal stem cell is derivatized to osteocyte.Usage factor method for designing result of study in 8 * 7 * 2 designs: cell component (7) * sample size (8) * body is implanted into the time (2).

As above assay.In addition, carry out 2 weeks and 1 week before the evaluation of follow-up new formation skull in the book office dead time, lumbar injection calcein and alizarin (people such as Parfitt, 1987; Kopher and Mao, 2003; People such as Clark, 2005).Data analysis and statistics are as mentioned above.In addition, utilize quantitative bone formation speed of dynamic organization's surveying (BFR) and mineralising engaging speed (MAR) to carry out quantitatively (people such as Parfitt, 1987 by fluorescence microscope; Kopher and Mao, 2003; People such as Clark, 2005).

The twin long factor of being sent can be not only also has compound influence to the cell line of being sent and to the host cell of invading in the braincap environment.For example, except promoting angiogenesis, PDGF promotes propagation people such as (, 2000) Park of osteoprogenitor cells.The system of this complexity is necessary for the participation instrument that the braincap defective that can not repair significant dimensions is provided.The dosage of the twin long factor (herein being VEGF and PDGF) although optimize among the embodiment 3 in the above, but still may need to modify according to the endogenous somatomedin that may exist in the braincap defect model.

Embodiment 9: separate and amplification cultivation bone marrow derived hematopoietic stem cell and mescenchymal stem cell.

According to described in the top research with our the previous method of developing, separate bone marrow derived hematopoietic stem cell and mescenchymal stem cell (referring to for example, Alhadlaq and Mao, 2003; People such as Alhadlaq, 2004; People such as Alhadlaq, 2005; Stosich and Mao, 2005; People such as Marion, 2005; People such as Yourek, 2005; People such as Moioli, 2006; Marion and Mao, 2006; People such as Stosich, 2006).As work (people such as Alhadlaq, 2005 formerly; People such as Marion, 2005; People such as Yourek, 2005), obtain the bone marrow sample (AllCells that anonymous adult contributes by the commercial channel, Berkeley, CA) part with every kind of bone marrow sample is used to use RosetteSep test kit (AllCells, Berkeley, negative selection technical point CA) is sowed discord mesenchymal stem cells (hMSC).Use Dulbecco ' s ModifiedEagle ' s Medium-Low Glucose (DMEM-LG; Sigma, St.Louis, MO) the isolated M SC of amplification cultivation institute, wherein said DMEM-LG are replenished 10% hyclone (FBS) (Biocell, Rancho Dominguez, CA) and 1% antibiotic (1 * antibiotic-antifungal contains the penicillin G sodium of 100 units/ml, the amphotericin B (Gibco of the streptomycin sulfate of 100 μ g/ml and 0.25 μ g/ml, Invitrogen, Carlsbad, CA) (people such as Alhadlaq, 2005; People such as Marion, 2005; People such as Yourek, 2005, people such as Moioli 2005; People such as Stosich, 2006).Each bone marrow sample, the hMSC amplification is no more than 3 and substitutes in each experiment.According to previous experience, necessary hardly the amplification for 3～5 generations.Cultivation is at 95% air/5%CO ₂In hatch under 37 ℃.

Each donor utilizes identical bone marrow sample to come isolating hematopoietic stem cells.The CD34 antibody that use is connected on the magnetic bead carries out positive selection (RosetteSep).The purifying cells of use flow cytometer is used for determining the percentage ratio of the separation of C D34 of institute (CD34+) positive cell.Also assess the vigor of cell by trypan blue exclusion method (Trypan Blue exclusion).From initial non-adherent cell separation of C D34+ cell.Then collect non-adherent cell by under 37 ℃, being incubated in the vinyl disc that 96 hole fibronectin apply 3 days with the IMDM (HSC growth medium) that adds 10%FBS, and separation of C D34+ cell people 1998 such as () Shi.Shift out non-adherent cell, and transfer in the new hole.Repeat this process twice, the suspension cell that will still retain is paved plate and is made on its flat board that adheres to the fibronectin coating afterwards.

Embodiment 10:HSC is divided into endothelium class cell, and MSC is divided into osteoblast class cell.

After converging, hHSC is transferred to 24 holes, 12 holes and the 6 hole tissue dishes that fibronectin applies continuously, and finally transfer to the Petri dish.HSC derive endotheliocyte class cell will continue the amplification.Preliminary data shows that these cells demonstrate endothelial cell morphology, and expresses some endothelial cell marker things (referring to for example, top Fig. 3).In addition, the hHSC significantly many a kind of endothelial cell marker thing-von willebrand's factors (vWF) of endotheliocyte expression ratio control cells of deriving.Use has the HSC growth medium that contains 10%FBS of endothelial cell differentiation fill-in (ECS), makes the attached cell differentiation of fibronectin, and wherein said ECS comprises VEGF (10ng/mL), bFGF (1ng/mL) and IGF-1 (2ng/mL).According to method before, utilize skeletonization to stimulate fill-in, MSC is divided into osteoblast class cell, and wherein said skeletonization stimulates fill-in to contain 100nM dexamethasone, 50 μ g/ml ascorbic acid and 100mM β-phosphoglycerol (referring to for example, Alhadlaq and Mao, 2003; People such as Alhadlaq, 2004; People such as Alhadlaq, 2005; Stosich and Mao, 2005; People such as Marion, 2005; People such as Yourek, 2005; People such as Moioli, 2006; Marion and Mao, 2006).

Embodiment 11: make PLGA microsphere and encapsulation PDGF.

Above following, these programs describe and the research described in people such as Moioli (2006).PLGA is biocompatibility and biodegradable poly-(L-lactic acid) and gathers (glycolic) synthetic copolymer, and be widely used (referring to for example, people such as Lu, 2000; People such as Shea, 2000; People such as Burdick, 2001; People such as Hedberg, 2003; People such as Karp, 2003a; People such as Ochi, 2003; People such as Moioli, 2006).(PLGA: 50: 50, PLA: PGA) (Sigma, St Louis Mo) were dissolved in the 1mL dichloroethanes for 250mg poly-(L-lactic acid) and poly-(glycolic) altogether.By as the work at us before described in two emulsifying technologies use PLGA microspheres encapsulate PDGF people such as (, 2006) Moioli.With this mixture whirlpool 1 minute.After adding 2ml 1%PVA, with mixture whirlpool 1 minute again.Resulting emulsion is joined in the 100ml 0.1% PVA solution.The mixture of PVA/ microsphere is joined in 100ml 2% isopropyl alcohol removing dichloroethanes, and the sclerosis microsphere, and under chemical ventilated chamber continuous stirring 2 hours.By filter collecting the PDGF microsphere, and then lyophilization, then be dissolved in the chloroform 4 hours, then acutely rocked 2 minutes.After clarification 4 hours, use PDGFELISA test kit (R﹠amp; D Systems, St.Louis MO), carries out quantitatively the concentration of the packaged PDGF of per unit microsphere according to the product rules.The microsphere that encapsulates the PDGF of scheduled volume is suspended among the 10ul PBS.Behind the inoculating cell, before implanting, the PDGF that the PLGA microsphere is encapsulated is expelled in the Matrigel solution by microtip.

Embodiment 12: the construct of perfusion inoculating cell

If cell survival is poor in the β TCP construct of Matrigel infusion, then can transmit (people such as Vunjak-Novakovic, 1999 by the perfusion bioreactor enhancing substance of exploitation in formerly working; 2002).In brief, set up the perfusion cultures base by the support in 10～100 mu m ranges with linear speed, this is corresponding to the rate of flooding of nature bone.In each passage, externally annular gas exchanger oxygen and pH aspect, culture medium keeps balance.Speed with every day 50% is changed culture medium.According to the listed engineering vascularization bone result of top table 3, infusion time is optimized.

Embodiment 13: form the braincap defective of complete thickness and the operation of engineering construct and implant.

The nude mice that 11 weeks are big contains 90% gram by lumbar injection (IP), and he orders (100mg/ml; Aveco, Fort Dodge is IA) with 10% xylazine (20mg/ml; Mobay, Shawnee, mixture anesthesia KS).Povidone iodine (10%) is used for operative region is carried out disinfection.Cut the long linear cut of 3cm to the midline along the footpath of skull.Push subcutaneous tissue and periosteum aside, expose brain cortical bone surface.According to employed method before, use the aseptic dental handpiece of phosphate-buffered saline flushing, form the braincap defective (5 * 1mm of through thickness in the central authorities of parietal bone ³: diameter 5mm) (referring to for example, Hong and Mao, 2004; People such as Moioli, 2006).According to previous experience, the braincap defective of this 5mm diameter through thickness has constituted important defective, do not transplant by bone and it can't be repaired (referring to for example, Hong and Mao, 2004; People such as Moioli, 2006).Cerebral dura mater and contiguous epicranial suture keep intact (Kopher and Mao, 2003; Hong and Mao, 2004; People such as Moioli, 2006).As in above-mentioned research, under 4 ℃ of slight decompressions, HSC or the HSC endotheliocyte of deriving is seeded in the aqueous phase of Matrigel.Matrigel is the basement membrane macromolecule hydrogel, its be widely used in inner skin cell viscosity echo angiogenesis experiment (referring to for example, people such as Abilez, 2006; People such as Baker, 2006; People such as Bruno, 2006; People such as Mondrinos, 2006; People such as Rajashekhar, 2006).Cell-Matrigel solution is infused into by inoculation hMSC derives in the hole of osteoblastic β TCP dish, then form gel down at 37 ℃.β TCP obtains by the commercial channel, its aperture between 200～400 μ m (BD BioScience, San Diego, CA).Engineered tissue construct with β TCP support will be fit to the braincap defective of 5mm diameter through thickness, use the common internal organs of 4-0 can absorb operation suture thread (plain gut absorbable surgical suture) suture operation tissue subsequently, this operation tissue is made of periosteum, sub-dermal soft tissue and skin.

Embodiment 14: organize results, histology and immunohistochemistry.

The braincap sample of being gathered in the crops that contains the engineering bone is used to carry out the demineralization prepared product, and being used for paraffin embedding, and demineralization is not embedded in the plastics, to the two fluorescently-labeled quantitative osseous tissue morphologys (calcein and alizarin) of using of new formation bone.For the demineralization prepared product, in sample stuck-at-0% paraformaldehyde, demineralization in isopyknic 20% sodium citrate and 50% formic acid is embedded in the paraffin, use as above-mentioned research in the normal structure program with the thickness transverse plane of 10 μ m cut into slices (referring to people such as Mao, 1998; Wang and Mao, 2002; People such as Kopher, 2003).Use hematoxylin and eosin, Feng Kusa dyeing and Masson three dyeing to carry out showing one's color of various zones in the engineering bone successive section.The prepared product of demineralization is not following described.The method that the immunohistochemistry of skeletonization and angiogenesis label is developed before following (referring to for example, Alhadlaq and Mao, 2005; People such as Stosich, 2006; Sundaramurthy and Mao, 2006).

Embodiment 15: by the quantitative bone geometry of computer organization norphometry.

By the computer organization norphometry separate to the engineering bone carry out quantitative testing (ImageProand Nikon Eclipse E800, Nikon Corp., Melville, NY).The grid of structure standard (1175 * 880 μ m ²) and under 4 times of object lens, place on the tissue sample, so that can carry out quantitatively to the engineering bone.Numerical data is carried out as the statistical analysis described at each embodiment.

Embodiment 16: by two fluorescent labelinies and computer aided dynamic osseous tissue norphometry new formation cranium is carried out quantitatively.

2 weeks and 1 week before execution, with calcein green (15mg/kg) and alizarin red (20mg/kg) lumbar injection, and by computer aided dynamic osseous tissue norphometry show one's color (people such as Parfitt, 1997; Mao, 2002; Kopher and Mao, 2003; People such as Clark, 2005).In gradient ethanol and acetone, the braincap sample is repaired (trim) and dehydration, further use the preparation of 85% methyl methacrylate (MMA) and 15% dibutyl phthalate to be used for not deliming embedding.Use band saw (band saw) that polymeric MMA-sample blocks is repaired.Use can cut not that the Leica of calcified tissue's sample of demineralization cuts ultramicrotome more, cuts the not 15-μ m section of demineralization continuously.Under fluorescence microscope to not carried out imaging (Mao, 2002 by the new mineralising bone of calcein labelling in the section of demineralization; Kopher and Mao, 2003; People such as Clark, 2005).Calculate mineralising engaging speed (MAR) (people such as Clark, 2005) by the average distance of measuring between follow-up calcein and the alizarin labelling divided by the interval (7 days) of injection of labelled.Bone formation speed (BFR) is defined as bone formation speed (BFR/BS) and is defined as MARX BSf/BS (people such as Clark, 2005).Numerical data is carried out as the statistical analysis described at each embodiment.

Embodiment 17: use atomic force microscope microscopical identification engineering bone

Mechanical performance by using the method testing engineering bone that atomic force microscope (AFM) sets up (referring to for example, people such as Hu, 2001; Patel and Mao, 2003; People such as Radhakrishnan, 2003; Allen and Mao, 2004; People such as Tomkoria, 2004; People such as Clark, 2005).The latter use the mechanical test of AFM more favourable, because can not distinguish the independent mechanical performance of engineering bone than macroscopical mechanical test.Use rapid draing cyanoacrylate rapid draing sample on microscope slide.Use two-sided tape, microscope slide is fixed on the rustless steel chassis of AFM, then magnetic force is installed on the piezoelectric scanner of AFM.In the process of AFM minute-pressure, sample continued the perfusion phosphate buffer.With the cantilever of nominal force constant k=0.12N/m and the Si of oxide sharpening ₃N ₄The tip is used for the construct surface of new results is applied minute-pressure.By in the Z plane, promoting cantilever tip acquisition power spectrum data.Record power distribution collection of illustrative plates is included in the data acquisition of minute-pressure load in the elongation of cantilever tip and the withdrawal process and corresponding displacement in the Z plane.Then compose data by following Hertz Model Calculation Young's modulus (E) from power, it has defined the relation between contact radius, minute-pressure load and the center displacement:

E＝3F(1-v)/4√Rδ ^3/2

Wherein, E is Young's modulus (Young ' s modulus), and F is the nano-machine load (nanomechanical load) that is applied, and v is the Poisson ratio (Poisson ' s ratio) of given area, R is the radius of curvature at AFM tip, and δ is the amount of impression (indentation).Determine the Young's modulus value of all group constructs, and compare with the similar value of the natural spongy bone that is obtained before.The average Young's modulus of diverse location is carried out statistical analysis to show their mechanical performance respectively.

Embodiment 18: use twin shaft MTS mechanical test equipment that the engineering bone is compressed with shear force character and carry out mechanical test.

Collect the all-work bone.Use PBS solution that collected sample is cleaned; absorb water fully to go out too much water; and use dental plaster to wrap up (potted) (Lab Buff; Miles Dental Products; South Bend is IN) to protect sample (MTS858 Mini Bionix II Machine, MTS Corp. in testing equipment; Minneapolis, MN).Initial load speed compressive load sample with 0.1mm/s.Ergometry (N) is than displacement (mm), and calculates the elastic modelling quantity of each sample, E (KPa).For shear force test, one of end of the bone of side parcel is connected on the bearing axle, other lateral parts is connected on the fixed rack (fixedstage).Shifting axle is applied initial low displacement (0.01mm/s), move active side with respect to stiff end.Use the resulting modulus of shearing of Station Manager software measurement.For compression and shear force load testing, all study different loading speeds to determine to the influence of mechanical test result to loading speed, if and loading speed influences the result, then use loading speed in the physical load scope of 1～4Hz people such as (, 2004) Collins.

Embodiment 19: use digital X-axis and μ CT that the engineering bone is carried out imaging.

(Faxitron, Wheeling IL) carry out imaging according to our disclosed approach (people 2005 such as Collins) to the engineering bone to use digital X-ray.In engineering bone stuck-at-0% formalin, and (ViVa CT 40, Scanco Switzerland) utilize multilamellar to carry out imaging with the resolution of 21 μ m to use microcomputer x-ray tomography (μ CT) system.Image reconstruction is become 5x5x1mm ³Volume, and determine marginal value according to bone pixel from image histogram and the peak-to-peak paddy of soft tissue pixel.Geometric widths to the engineering bone is carried out quantitatively.All numerical value all utilizes the ANOVA of Bonferroni check.Also the natural human font bone (lamboidal bone) of adjacency is carried out the contrast of imaging as the engineering bone by μ CT.To the analysis of the μ CT data of natural human font bone and engineering bone photo with.

Embodiment 20: major path and bFGF promote the neovascularization of host tissue.

Carry out and similar experiment described in embodiment 3, but use lower bFGF concentration.

To gather (MW3400 in (ethylene glycol) diacrylate (PEGDA); Nektar, Huntsville, AL USA) is dissolved among the PBS (6.6%w/v), additional 133 units/mL penicillin and 133mg/mL streptomycin (Invitrogen, Carlsbad, CA, USA).Concentration with 50mg/mL adds light trigger 2-hydroxyl-1-[4-(hydroxyl-oxethyl) phenyl]-2-methyl isophthalic acid-acetone (Ciba, Tarrytown, NY, USA).The UV light that uses 365nm to resulting PEG cylinder carried out photopolymerization 5 minutes (Glo-Mark, Upper Saddle River, NJ, USA).Make 3 kinds of PEG hydrogel structures altogether: 1) at photopolymerization PEG hydrogel middle punch 3 major path (diameters: 1mm) (referring to for example Figure 15 A) altogether; 2) 0.5 μ g/ μ L bFGF is loaded in the PEG hydrogel, and does not have major path (referring to for example Figure 15 B); And 3) combination of 0.5 μ g/ μ L bFGF and major path (referring to for example Figure 15 C).

(the C.B17 strain of immunodeficiency (SCID) male mice is made up in strictness; Mus 4-5 in age week) restrains his life (100mg/kg) and xylazine (4mg/kg) anesthesia by lumbar injection.Build the hair of mouse back, and place prone position, then use 10% povidone iodine and 70% ethanol to carry out disinfection.The long line style otch of tangent line (upper midsagittal line) excision 1cm then carries out blunt dissection to form subcutaneous pouch on the back.Every SCID mice is accepted three kinds of PEG hydrogel implants: have major path but do not have the PEG of bFGF, load bFGF but do not have the PEG of major path perhaps has the two PEG of bFGF and major path.Use common internal organs can absorb 4-0 suture otch.Implant all 4 weeks of PEG hydrogel cylinder in vivo.

After subcutaneous 4 weeks of implantation, gather in the crops the PEG hydrogel sample at the SCID mouse back.。At CO ₂After suffocating, the otch sterilely at the back of SCID mice.After shifting out on every side fibrous capsule carefully, separate PEG hydrogel cylinder from the host, use PBS to clean, and in 10% formalin, fix 24 hours.Then the PEG sample is embedded in the paraffin, and with the transverse plane of 5 μ m thickness cut into slices (cross-section major path is referring to Figure 15 A).Use hematoxylin and eosin that paraffin section is dyeed.Prepare successive adjacent slices and be used for immunohistochemistry.Deparaffnize is carried out in section, in PBS, clean, at room temperature use bull testis hyaluronidase (1600U/ml) in having the pH5.5 sodium-acetate buffer of 150mM sodium chloride, to digest 30 minutes.All immunohistochemistry programs are followed method before us (people such as Mao, 1998; Alhadlaq and Mao, 2005; Sundaramurthy and Mao, 2006).In brief, at room temperature use 5% bovine serum albumin (BSA) to slicing treatment 20 minutes, with the blocking-up nonspecific reaction.Use following antibody: the anti-vascular endothelial growth factor factor (anti-VEGF) (ABcam, Cambridge, MA USA) and biotin labeled lectins (WGA) from Radix Betae (tritium vulgaris) (tritin), has or do not have its inhibitor acetylurea acid (actyleuraminic acid) (Sigma, St.Louis, MI, USA).WGA is in conjunction with the glycosyl that is rich in the vascular endothelial cell of α-D-GlcNAc and NeuAc (people such as Jinga, 2000; People such as Izumi, 2003).In humidity chamber with the primary antibody overnight incubation after, use PBS to clean section, and with the secondary antibody (1:500 of anti-mice IgG; Antibodies Inc., Davis CA) is hatched 30 minutes.Then, will cut into slices and in humidity chamber, hatch 30 minutes with streptomycin-HRP conjugate.After in PBS, cleaning, reuse the dual link program of secondary antibody.Use diaminobenzadine (DAB) solution to produce slide, and used Mayer ' s haematoxylin redyeing color 3～5 minutes.The slide of counterstain dewaters in gradient ethanol, and cleans in dimethylbenzene.Except that saving one-level antibody, negative control is carried out identical program.

The result shows, after SCID mouse back body was implanted into for 4 weeks, has major path but do not have the acellular PEG hydrogel of bFGF to show the host tissue infiltration, and it is only in the inner chamber of major path, and not in other positions of PEG (referring to for example Figure 15 A ').On the contrary, load bFGF but do not have the acellular PEG hydrogel of major path obviously to have at random and the zone (referring to for example Figure 15 B ') of host tissue infiltration independently.Have that the two PEG hydrogel of major path and bFGF shows then that host tissue inwardly is grown in the major path and not in other positions of PEG (referring to for example Figure 15 C ').The two PEG hydrogel that all lacks of major path and bFGF shows does not have host tissue infiltration (not video data), and this lacks host tissue penetrates into the PEG hydrogel from host cell data consistent (people such as Alhadlaq, 2005 with showing before; Stosich and Mao, 2005; 2006).

Embodiment 21: separate and amplification cultivation people bone marrow derived mescenchymal stem cell (hMSC)

Consistent with program listed in embodiment 9, separate and amplification cultivation people bone marrow derived mescenchymal stem cell (hMSC).

According to before method (referring to for example, people such as Marion, 2005; People such as Yourek, 2005; People such as Moioli, 2006; Marion and Mao, 2006), from the fresh bone marrow sample separation people MSC of two anonymous adult donors (AllCells, Berkeley, CA).Behind the 50mL pipe, (StemCell Technologies, Vancouver Canada), and were at room temperature hatched 30 minutes to add altogether 750 μ L RosetteSep with the bone marrow sample transfer.Then add the PBS of 15mL in 2%FBS and 1mM EDTA solution in the bone marrow sample, reaching cumulative volume is about 30ml.Then with the bone marrow sample at 15mL Ficoll-Paque (StemCell Technologies) higher slice, and centrifugal 25 minutes of 3000g at room temperature.The cellular layer of whole enrichment is taken out from the Ficoll-Paque interface.With mixture centrifugal 10 minutes with 1000rpm.Solution is drawn into 500 μ L Dulbecco ' s Modified Eagle ' s Medium (Sigma-Aldrich Inc.St.Louis, MO), wherein Dulbecco ' s Modified Eagle ' s Medium has replenished 10% hyclone (FBS) (Atlanta Biologicals, Lawrenceville, GA) and 1% antibiont-antifungal (Gibco, Carlsbad CA), is called basal medium with it in the back.To isolating monocyte count, according to about every 100mmPetri dish 0.5～1 * 10 ⁶Individual cell is paved plate, and at the total 37 ℃ of following 5%CO of basal medium ₂In hatch.24 as a child, removes non-adherent cell, and use phosphate buffer (PBS) to clean twice, hatches 12 days (25) by every other day changing fresh culture.90% converge after, use 0.25% trypsin and 1mMEDTA to take out cell from flat board in following 5 minutes, and be layered on again in the 100mmPetri dish at 37 ℃, be known as 1 generation cell.

Embodiment 22: human mesenchymal stem cell is divided into living lipocyte.

According to before method (referring to for example, people such as Alhadlaq, 2005; Stosich and Mao, 2005,2006; Marion and Mao, 2006), by being exposed to into the fat culture medium, secondary and three generations hMSC is induced to be divided into living lipocyte, and wherein said one-tenth fat culture medium is made of the basal medium that is replenished 0.5 μ M dexamethasone, 0.5 μ M isobutyl methylxanthine (IBMX) and 50 μ M indomethacins.The subgroup of continuous culture hMSC in basal medium also is at 95% air and 5%CO ₂In under 37 ℃, every other day change culture medium.(Sigma-Aldrich, St.Louis MO) confirm lipogenesis (lipogenesis) to use oil red O stain.For external check lipogenesis differentiation, use into fat culture medium processing hMSC and be up to for 5 weeks.To use or not use in monolayer culture hMSC stuck-at-0% formalin of lipogenesis differentiation, and carry out oil red O stain.Detect existing of dull and stereotyped fat bubble with the 10X amplification or lack at anti-phase microscopically.

The result shows 35 days of process isolated culture, and human mesenchymal stem cell becomes to give birth to lipocyte (referring to for example Figure 16) in vitro differentiation.Compare (referring to for example Figure 16 A-17E) with the hMSC that does not have lipogenesis differentiation, hMSC derive living lipocyte and oil red O stain positive reaction, and through increasing (referring to for example Figure 16 F-17J) in 35 days day by day.After this and before data consistent, data show were before handled in becoming the fat culture medium and be lower than for 2 weeks, the hMSC living lipocyte of deriving was expressed PPAR-γ 2 (referring to for example, people such as Alhadlaq, 2005).Through observed 35 days, to derive between the living lipocyte at hMSC and hMSC, total dna content of culture sample lacks statistically significant difference.Yet at the 28th day and 35 days that cultivates, hMSC derived the glycerol content of living lipocyte significantly than hMSC height, and this hint hMSC living lipocyte of deriving assembles progressively external that fat steeps in the cell.

Embodiment 23: derive living lipocyte and body of encapsulation hMSC is implanted in the PEG hydrogel.

In the parallel laboratory test that utilizes the above-mentioned model system that has major path and bioactie agent in the PEG hydrogel (referring to embodiment 3), hMSC and the hMSC living lipocyte of deriving is packed to determine whether that engineering major path and bFGF promote the vascularization lipogenesis.

The PEG hydrogel is dissolved among the aseptic PBS, and (Gibco, Carlsbad CA) reach the final solution of 10w/v% to replenish 100U/ml penicillin and 100 μ g/ml streptomycins.Light trigger 2-hydroxyl-1-[4-(hydroxyl-oxethyl) phenyl]-(NY USA) joins in the PEGDA solution 2-methyl isophthalic acid-acetone for Ciba, Tarrytown.After in basal medium, becoming the fat differentiation or cultivating for 1 week, use 0.25% trypsin and 1mM EDTA to take out hMSC or the hMSC living lipocyte of deriving from culture plate in following 5 minutes at 37 ℃, and with 3 * 10 ⁶The density of individual cell/mL is resuspended in respectively in PEG polymer/photoinitiator solution.A 75 μ l cell/polymer/light trigger suspensions are loaded into (Fisher Scientific in the aseptic plastic medicated cap of 0.075mL micro-centrifuge tube (6 * 4mm: diameter * highly), Hampton, NH), then use long wave 365nm uviol lamp (Glo-Mark, Upper Saddle River, NJ) about 4mW/cm ²Intensity carried out photopolymerization 5 minutes.Take out photopolymerisable cell-PEG construct from plastic cap, and transfer in the 12 hole flat boards of the fatty culture medium of corresponding one-tenth.Before photopolymerization, 0.5 μ g/ μ LbFGF loads in the PEG hydrogel altogether.Form 3 major paths (referring to embodiment 20) according to approach recited above.After subcutaneous 12 weeks of implantation, gather in the crops PEG hydrogel cylinder at the back of nude mouse.All organized processing, histology and immunohistochemistry program (referring to embodiment 20) same as described above.

The result shows that the PEG hydrogel does not allow Premeabilisation of cells (referring to for example Figure 18 A ').These discoveries consistent with research before (referring to for example, people such as Alhadlaq, 2005; Stosich and Mao, 2005; 2006).Yet the PEG hydrogel of load institute through engineering approaches major path and bFGF not only shows darker color, and has 3 red circles (referring to for example Figure 18 B ') at transverse plane.Further, have major path and bFGF the two and shown that by the derive PEG hydrogel of living lipocyte of inoculation hMSC darker color is not only arranged, and also have red circle (referring to for example Figure 18 C).After the check of histology and immunohistochemistry, the derive major path of living lipocyte and structure and the PEG hydrogel of bFGF of encapsulation hMSC shown formative tissue island (referring to for example Figure 19 A).As shown in Figure 19 B, many engineered tissues island is that oil red is male, and there is lipogenesis in this hint.VEGF antibody is obviously showing positive staining (referring to for example Figure 19 C) in the stroma, and anti-WGA agglutinin antibody is positioned at neighbouring (referring to for example Figure 19 D) of engineering fatty tissue, and this hint engineering new vessels can promote lipogenesis.

Embodiment 24: the molecular marker of vascular endothelial cell

The expression of analyzed VEGF 2 of blood vessel CFU-GM or Flk1, the two is the molecular marked compound of vascular endothelial cell.Consistent with described in embodiment 2 carries out hematopoietic stem cell separation, cultivation, differentiation and labelling.

The result show with buffer solution (buffer sulocation) not VEGF expression/Flk1 compare, the blood vessel CFU-GM (in first row 1 generation cell and the 2nd row in 2 generation cell) be found and express vascular endothelial cell growth factor 2 or Flk1 the two (referring to for example Figure 20).The quantitative demonstration P1 of VEGF2 content and the significantly many VEGF2 (referring to for example Figure 21) of the equal expression ratio buffering of P2 cell substrate.

These digital proofs as by two kinds of endothelial cell marker thing VEGF and Flk1 expression proved, can be divided into endotheliocyte class cell from the isolating HSC of people's bone marrow.

Embodiment 25: the cell marking experiment.

To being analyzed, occupy in the time of two kinds of cell types by the porous β TCP support of inoculation osteoprogenitor cells and blood vessel CFU-GM.The method of support infusion CFU-GM is consistent with embodiment 1 described method.

The result shows that the osteoprogenitor cells of green fluorescent protein (GFP) labelling and the blood vessel CFU-GM of red CM-Dil labelling occupy simultaneously in porous β TCP support (referring to for example Figure 22).As mentioned above, the β TCP support that body is implanted into inoculation osteoprogenitor cells and blood vessel CFU-GM has produced formation vascularization bone, and (referring to for example, embodiment 1; Fig. 2).

These digital proofs by co-inoculation in bio-compatible material different spaces zone people's osteoprogenitor cells and the blood vessel CFU-GM when occupying support jointly, can successfully break up skeletonization and vascular tissue respectively.

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Claims

1. organization component, it comprises:

(a) biocompatible matrix;

(b) blood vessel CFU-GM; With

(c) tissue CFU-GM;

Wherein said assembly exsomatizes, perhaps (a) and (b) or (c) at least a be allogenic for the vertebrates receptor.

2. form the method for blood vessel tissue's assembly, it comprises:

With (a) biocompatible matrix, (b) tissue CFU-GM and (c) blood vessel CFU-GM combination;

Form substrate like this, organized the combination of CFU-GM and blood vessel CFU-GM.

3. the method for claim 2, it also comprises the combination of hatching substrate, organizing CFU-GM and blood vessel CFU-GM.

4. each method of claim 2-3, wherein said combination are to carry out exsomatizing.

5. each method of claim 3-4 wherein saidly hatches that comprising exsomatizes hatches.

6. each method of claim 3-5, wherein said hatching comprises in the body and hatching.

7. each assembly or method of claim 1-6, wherein said to organize CFU-GM to be selected from following: mescenchymal stem cell (MSC), MSC derived cell, osteoblast, chondrocyte, myocyte, adipose cell, neuronal cell, neurogliocyte, fibroblast, myocardial cell, hepatocyte, nephrocyte, bladder cell, β-islet cells, odontoblast, pulp cells, periodontal cell, tendon cell, pneumonocyte, heart cell and combination thereof.

8. the assembly of claim 7 or method, wherein said fibroblast is selected from following: a matter fibroblast, tendon fibroblast, ligament fibroblast, periodontal fibroblast and cranium face fibroblast.

9. each assembly or method of claim 1-8, the wherein said CFU-GM of organizing is the MSC chondrocyte.

10. each assembly or method of claim 1-9, the wherein said CFU-GM of organizing is MSC.

11. each assembly or method of claim 1-10, wherein said blood vessel CFU-GM is selected from following: hematopoietic stem cell (HSC), HSC endotheliocyte, vascular endothelial cell, lymphatic endothelial cells, cultivation endotheliocyte, the former foster endotheliocyte of being commissioned to train, bone marrow stem cell, notochord cell, Human umbilical vein endothelial cells (HUVEC), lymph gland endotheliocyte, endothelial progenitor cells, the stem cell that is divided into endotheliocyte, smooth muscle cell, matter fibroblast and myofibroblast.

12. each assembly or method of claim 1-11, wherein said blood vessel CFU-GM is HSC.

13. each assembly or method of claim 1-11, wherein said blood vessel CFU-GM is the HSC endotheliocyte.

14. comprising, each assembly or method of claim 1-13, wherein said substrate be selected from following material: fibrin, Fibrinogen, collagen, poe, polyvinyl alcohol, polyamide, Merlon, polyvinylpyrrolidone, ocean attachment proteins, cyanoacrylate, macromolecule hydrogel and its combination.

15. each assembly or method of claim 1-14, wherein said substrate comprises macromolecule hydrogel.

16. each assembly or method of claim 1-15, wherein said substrate comprises at least one physical channel.

17. the organization component of claim 16 or method, wherein said substrate comprise that a plurality of average diameters are that about at least 0.1mm is to the physical channel that is up to about 50mm.

18. the organization component of claim 17 or method, wherein said a plurality of physical channels have following average diameter: about 0.2mm, about 0.3mm, about 0.4mm, about 0.5mm, about 0.6mm, about 0.7mm, about 0.8mm, about 0.9mm, about 1.0mm, about 1.1mm, about 1.2mm, about 1.3mm, about 1.4mm, about 1.5mm, about 1.6mm, about 1.7mm, about 1.8mm, about 1.9mm, about 2.0mm, about 2.5mm, about 3.0mm, about 3.5mm, about 4.0mm, about 4.5mm, about 5.0mm, about 5.5mm, about 6.0mm, about 6.5mm, about 7.0mm, about 7.5mm, about 8.0mm, about 8.5mm, about 9.0mm, about 9.5mm, about 10mm, about 15mm, about 20mm, about 25mm, about 30mm, about 35mm, approximately 40mm or approximately 45mm.

19. each assembly or method of claim 1-18, wherein said substrate also comprises somatomedin or somatomedin is incorporated into the step of described host material.

20. the assembly of claim 18 or method, wherein said somatomedin is an angiogenesis growth factor.

21. each assembly or method of claim 19-20, wherein said somatomedin is selected from bFGF, VEGF, PDGF, TGFb and its combination.

22. each assembly or method of claim 1-21, wherein said assembly comprises CFU-GM, and the density of described CFU-GM is about at least 0.0001 * 1,000,000 cell (M) ml ^-1To being up to about 1000M ml ^-1

23. the assembly of claim 22 or method, wherein said CFU-GM appears in the substrate with following density: about 1M ml ^-1, about 5M ml ^-1, about 10M ml ^-1, about 15M ml ^-1, about 20M ml ^-1, about 25M ml ^-1, about 30M ml ^-1, about 35M ml ^-1, about 40M ml ^-1, about 45M ml ^-1, about 50M ml ^-1, about 55M ml ^-1, about 60M ml ^-1, about 65M ml ^-1, about 70M ml ^-1, about 75M ml ^-1, about 80Mml ^-1, about 85M ml ^-1, about 90M ml ^-1, about 95M ml ^-1Or about 100M ml ^-1

24. each assembly or method of claim 1-23, its medium vessels CFU-GM is that about 100:1 is to being up to 1:100 with organizing the ratio of CFU-GM.

25. the assembly of claim 24 or method, its medium vessels CFU-GM is about 20:1 with organizing the ratio of CFU-GM, about 19:1, about 18:1, about 17:1, about 16:1, about 15:1, about 14:1, about 13:1, about 12:1, about 11:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15, about 1:16, about 1:17, about 1:18, approximately 1:19 or approximately 1:20.

26. the method for treated tissue or organ defective, it comprises is transplanted to each assembly of claim 1 or 6-15 in the individuality of these needs.

27. identify the method for the candidate molecules that improves tissue blood vesselization, it comprises:

With substrate, organize each organization component of CFU-GM, blood vessel CFU-GM, its combination or claim 1 or 7-26 to contact with candidate molecules;

Hatch described organization component; And

Measure the vascularization of described organization component.

28. the method for claim 27, it also comprises mensuration with respect to the control tissue assembly that does not contact with candidate molecules, and in described organization component, whether vascularization improves.

29. identify the method for the candidate molecules that reduces tissue blood vesselization, it comprises:

Hatch described organization component; And

Measure the vascularization of described organization component.

30. according to the method for claim 29, it also comprises mensuration with respect to the control tissue assembly that does not contact with candidate molecules, in described organization component, whether vascularization reduces.

31. each assembly or method of claim 1-30, wherein said organization component is bone, fat, bladder, brain, breast, bone cartilage junction, central nervous system, spinal cord, peripheral nervous, neuroglia, esophagus, fallopian tube, heart, pancreas, intestinal, gallbladder, kidney, liver, lung, ovary, prostate, spleen, skeletal muscle, skin, stomach, testis, thymus, thyroid, trachea, urogenital tract, ureter, urethra, a matter soft tissue, periosteum, periodontal tissue, sutura cranii, hair follicle, oral mucosa or uterine cancer cell assembly.

32. each assembly or method of claim 1-31, wherein said organization component is the osseous tissue assembly.

33. each assembly or method of claim 1-31, wherein said organization component is the fatty tissue assembly.