CN101500508A - Biomolecule-linked biomimetic scaffolds - Google Patents
Biomolecule-linked biomimetic scaffolds Download PDFInfo
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- CN101500508A CN101500508A CNA2007800292629A CN200780029262A CN101500508A CN 101500508 A CN101500508 A CN 101500508A CN A2007800292629 A CNA2007800292629 A CN A2007800292629A CN 200780029262 A CN200780029262 A CN 200780029262A CN 101500508 A CN101500508 A CN 101500508A
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Abstract
The invention provides a composition comprising a nanofiber polymer in which the fibers of the nanofiber polymer are aligned, and a molecule is covalently attached, either directly or through a linker, to the nanofiber polymer. This molecule is capable of either covalently or non-covalently attaching to a member selected from an extracellular matrix component, a growth factor, and combinations thereof. The invention also provides methods of making the composition and methods of using the compositions to add new tissue to a subject, such as a human.
Description
The cross reference of related application
[0001] the application is the part continuation application of Application No. 11/668,448, and requires U.S. Provisional Patent Application serial number 60/______ (the Attorney Docket No of submission on June 5th, 2007.061818-02-5053 PR01), the U.S. Provisional Patent Application No that submit to 2006 on November 30.The No that on June 9th, 60/861,780,2006 submitted to.60/804,350 priority, its this by reference integral body incorporate this paper into and be used for all purposes.
Thanks for government-funded
[0002] subsidy of the application's national health committee, government-funded project appropriation (contract) number is HL078534.There is certain right in government for the present invention.
Background of invention
[0003] this area needs to replace or to improve the compositions of experimenter's biological function.This area also needs to promote the compositions of experimenter's growth of new tissue or replacing damaged tissue.Invention described herein has solved these and other needs.
Summary of the invention
[0004] in first aspect, the invention provides the compositions that contains the first cellulosic polymers support and biomolecule, wherein said biomolecule is non-covalent or be covalently attached to the described first cellulosic polymers support directly or by junctional complex.In exemplary, described biomolecule is an anti-platelet agents.In an exemplary embodiment, one or more fiber of the described first cellulosic polymers support is aligned.In an exemplary embodiment, wherein the first cellulosic polymers support has the length that is selected from following scope: about 0.01cm is to about 20cm, about 0.05cm is to about 5cm, about 0.5cm is to about 5cm, about 1cm is to about 5cm, about 2cm is to about 5cm, and about 1cm is to about 3cm, and about 2cm arrives about 15cm to about 10cm and about 5cm.In another exemplary embodiment, said composition has the shape that is selected from following shape: thin slice, pipe is filled pipe and bar.In another exemplary embodiment, said composition has the pipe of being selected from, and fills the shape in pipe and the bar.In another exemplary embodiment, said composition is shaft-like.In another exemplary embodiment, described compositions has pipe or fills the shape of pipe.In another exemplary embodiment, the described first cellulosic polymers support is basically along being selected from vertically and the arrangement of the direction of circumference.In another exemplary embodiment, the first cellulosic polymers support has seam.In another exemplary embodiment, the first cellulosic polymers support is seamless.In another exemplary embodiment, first cellulosic polymers is integrally formed.
[0005] in another exemplary embodiment, be selected from following polymer or subunit at least a the comprising in the fiber of the first cellulosic polymers support: aliphatic polyester, polyalkylene oxide, polydimethylsiloxane, polycaprolactone, polylysine, collagen, laminin, fibronectin, elastin laminin, alginate, fibrin, hyaluronic acid, Dan Baijutang, polypeptide and combination thereof.In another exemplary embodiment, this aliphatic polyester is selected from as follows: lactic acid (D-or L-), lactide, poly-(lactic acid), poly-(lactide), hydroxyacetic acid, poly-(hydroxyacetic acid), poly-(Acetic acid, hydroxy-, bimol. cyclic ester), Acetic acid, hydroxy-, bimol. cyclic ester, poly-(lactide-copolymerization-Acetic acid, hydroxy-, bimol. cyclic ester), poly-(lactic acid-copolymerization-hydroxyacetic acid) and combination thereof.In another exemplary embodiment, at least a of the first scaffold fibers polymer fiber comprises poly-(lactide-copolymerization-Acetic acid, hydroxy-, bimol. cyclic ester) (PLGA).
[0006] in an exemplary embodiment, anti-platelet agents is to be selected from following member: adenosine diphosphate (ADP) antagonist or P
2Y
12Antagonist, phosphodiesterase (PDE) inhibitor, adenosine reuptake inhibitor, vitamin K antagonist, heparin, hyparinoids from animal organs, direct thrombin inhibitor, glycoprotein I IB/III inhibitor, antithrombase and officinal salt, isomer, enantiomer, the polymorph that comprises amorphous form, solvate, hydrate, cocrystallization, complex, active metabolite, reactive derivative and trim, its prodrug etc.In another exemplary embodiment, anti-platelet agents is direct thrombin inhibitor.In another exemplary embodiment, anti-platelet agents is to be selected from following member: hirudin, bivalirudin, lepirudin 023 ludon, desirudin, argatroban, dabigatran, dabigatran ester, melagatran, Xi Meijia group, its prodrug and analog.In another exemplary embodiment, anti-platelet agents is to be selected from following member: hirudin, bivalirudin, lepirudin 023 ludon, desirudin, its prodrug and analog.In another exemplary embodiment, anti-platelet agents comprises the thrombin binding site.In another exemplary embodiment, anti-platelet agents comprises protein sequence, and it is to be selected from following member: FPRP and LYEEPIEEFDGN.In another exemplary embodiment, the first cellulosic polymers support is to be selected from following member: bar, pipe and filling pipe.
[0007] in another exemplary embodiment, described biomolecule or anti-platelet agents are not covalently bound to first cellulosic polymers by junctional complex.In another exemplary embodiment, the invention provides and have the compositions that is selected from following structural formula member:
A---X---biomolecule (I) or
A---X---anti-platelet agents (Ia)
Wherein A is the first cellulosic polymers support, and X is selected from following member: covalent bond, 0, S, C (O), C (O) O, C (O) S, C (O) NH, S (O), S (O)
2, S (O)
2NR*, C (O) NR*, NH or NR*, wherein R* is selected from following member: alkyl replacement or unsubstituted, that replace or unsubstituted assorted alkyl, cycloalkyl replacement or unsubstituted, Heterocyclylalkyl replacement or unsubstituted, that replace or unsubstituted aryl, heteroaryl replacement or unsubstituted.In another exemplary embodiment, anti-platelet agents is to be selected from following member: heparin, hirudin, bivalirudin, lepirudin 023 ludon, desirudin, argatroban, dabigatran, dabigatran ester, melagatran, Xi Meijia group, its prodrug and analog.In another exemplary embodiment, anti-platelet agents is to be selected from following member: hirudin, hirudin analog, bivalirudin, lepirudin 023 ludon or desirudin.In another exemplary embodiment, anti-platelet agents comprises the thrombin binding site.In another exemplary embodiment, anti-platelet agents comprises protein sequence, and it is to be selected from following member: FPRP and LYEEPIEEFDGN.In another exemplary embodiment, the first cellulosic polymers support is seamless.In another exemplary embodiment, the first cellulosic polymers support is integrally formed.
[0008] in another exemplary embodiment, described compositions comprises that at least one has the part that is selected from following structure:
Symbol wherein
The part that expression shows is connected to the point of the remainder of polymer, A
1It is the subunit of the first cellulosic polymers support.In another exemplary embodiment, A
1It is subunit by the polymerization formation of monomer/series monomers.In another exemplary embodiment, A
1Be to be selected from following member: one or more monomers described herein or subunit.In another exemplary embodiment, A
1Be to be selected from following member: aliphatic polyester, polyalkylene oxides, polydimethylsiloxane, polyvinyl alcohol, polylysine, collagen protein, laminin, fibronectin, elastin laminin, alginate, fibrin, hyaluronic acid, Dan Baijutang, polypeptide and combination thereof.In another exemplary embodiment, A
1It is functionalized lactide part.In another exemplary embodiment, described functionalized lactide part functionalised in pendant methyl.In another exemplary embodiment, X is derived from the complementary interaction group on the first fibroid support and the reaction of anti-platelet agents.Description for these complementary interaction groups is provided in this article.In another exemplary embodiment, anti-platelet agents is to be selected from following member: hirudin, hirudin analog, bivalirudin, lepirudin 023 ludon or desirudin.In another exemplary embodiment, anti-platelet agents comprises the thrombin binding site.In another exemplary embodiment, anti-platelet agents comprises protein sequence, and it is to be selected from following member: FPRP and LYEEPIEEFDGN.In another exemplary embodiment, the first cellulosic polymers support is seamless.In another exemplary embodiment, the first cellulosic polymers support is integrally formed.
[0009] in another exemplary embodiment, described compositions comprises that at least one has the part that structure is selected from following structural formula member:
Symbol wherein
The part that expression shows is connected to the point of the remainder of polymer, and X is selected from following member: covalent bond, O, S, C (O), C (O) O, C (O) S, C (O) NH, S (O), S (O)
2, S (O)
2NR*, C (O) NR*, NH or NR*, wherein R* is selected from following member: alkyl replacement or unsubstituted, that replace or unsubstituted assorted alkyl, cycloalkyl replacement or unsubstituted, Heterocyclylalkyl replacement or unsubstituted, that replace or unsubstituted aryl, heteroaryl replacement or unsubstituted.In another exemplary embodiment, X is selected from following member: C (O) NH and C (O) NR*.In another exemplary embodiment, anti-platelet agents is to be selected from following member: heparin, hirudin, bivalirudin, lepirudin 023 ludon, desirudin, argatroban, dabigatran, dabigatran ester, melagatran, Xi Meijia group, its prodrug and analog.In another exemplary embodiment, anti-platelet agents is to be selected from following member: hirudin, hirudin analog, bivalirudin, lepirudin 023 ludon or desirudin.In another exemplary embodiment, anti-platelet agents comprises the thrombin binding site.In another exemplary embodiment, anti-platelet agents comprises protein sequence, and it is to be selected from following member: FPRP and LYEEPIEEFDGN.In another exemplary embodiment, X is connected to the C-terminal of hirudin.In another exemplary embodiment, X is connected to the aminoacid of the C-terminal of hirudin.In another exemplary embodiment, X is connected to hirudin by the side chain of aspartic acid or glutamic acid part.In another exemplary embodiment, X is connected to hirudin by the side chain of aspartic acid part.In another exemplary embodiment, X is connected to the amino acid moiety of hirudin, and this part is to be selected from following member: the D5 of wild type peptide sequence, E8, E17, D33, E35, E43, D53, D55, E57, E58, E61, E62 and Q65.In another exemplary embodiment, the first cellulosic polymers support is seamless.In another exemplary embodiment, the first cellulosic polymers support is integrally formed.
[0010] in another exemplary embodiment, the invention provides and have the compositions that is selected from following structural formula member:
A---X---L---biomolecule (II)
A---X---L-anti-platelet agents (IIa)
Wherein A is the first cellulosic polymers support, and X is selected from following member: covalent bond, 0, S, C (O), C (O) O, C (O) S, C (O) NH, S (O), S (O)
2, S (O)
2NR*, C (O) NR*, NH or NR* wherein R* are selected from following member: alkyl replacement or unsubstituted, assorted alkyl replacement or unsubstituted, that replace or unsubstituted cycloalkyl, that replace or unsubstituted Heterocyclylalkyl, aryl replacement or unsubstituted, heteroaryl replacement or unsubstituted, L is a junctional complex, anti-platelet agents is to be selected from following member, includes but not limited to heparin, hirudin, bivalirudin, lepirudin 023 ludon, desirudin, argatroban, dabigatran, dabigatran ester (oral formulations), melagatran, Xi Meijia group and prodrug thereof.In another exemplary embodiment, A is the pipe of the first cellulosic polymers support tube or filling, and wherein one or more fiber of the first cellulosic polymers support is aligned.In another exemplary embodiment, L comprises and is selected from following member: polyphosphazene, poly-(vinyl alcohol), polyamide, Merlon, polyalkylene, polyacrylamide, poly alkylene glycol, polyalkylene oxides, polyalkylene terephthalates, polyvinylether, polyvinyl ester, polyvinyl halides, polyvinylpyrrolidone, polyglycolide, polysiloxanes, polyurethane, poly-(methyl methacrylate), poly-(ethyl methacrylate), poly-(butyl methacrylate), poly-(isobutyl methacrylate), poly-(N-Hexyl methacrylate), polymethylacrylic acid isodecyl ester, polylauryl methacrylate, polymethyl acid phenenyl ester, poly-(acrylic acid methyl ester .), the polyacrylic acid isopropyl ester, polyisobutyl acrylate, polyoctodecyl acrylate, polyethylene, polypropylene, poly-(ethylene glycol), poly-(oxirane), poly-(PETP), poly-(vinyl acetate), polrvinyl chloride, polystyrene, polyvinylpyrrolidone, Pluronic, polyvinyl phenol, saccharide (glucosan for example, amylose, hyaluronic acid, poly-(sialic acid), heparan, heparin, or the like); Poly-(aminoacid), for example poly-(aspartic acid) and poly-(glutamic acid); Nucleic acid and copolymer thereof.In another exemplary embodiment, L comprises and is selected from following member: peptide, saccharide, poly-(ether), poly-(amine), poly-(carboxylic acid), poly-(aklylene glycol), such as poly-(ethylene glycol) (＂ PEG ＂), poly-(propylene glycol) (＂ PPG ＂), copolymer of ethylene glycol and propylene glycol or the like, poly-(oxyethylation polyhydric alcohol), poly-(enol), poly-(vinylpyrrolidone), poly-(hydroxypropyl methyl acrylamide), poly-(alpha-hydroxy acid), poly-(vinyl alcohol), poly-phosphorus piperazine, poly-oxazoline, poly-(N-acryloyl morpholine), Polysialic acid, polyglutamate, polyaspartic acid salts, polylysine, polymine, biodegradable polymer (polyactide for example, polyglycerin ester and copolymer thereof), polyacrylic acid.In another exemplary embodiment, L is selected from following member: Polyethylene Glycol and polypropylene glycol.In another exemplary embodiment, X is N, and L is selected from following member: Polyethylene Glycol and polypropylene glycol.In another exemplary embodiment, anti-platelet agents is to be selected from following member: hirudin, hirudin analog, bivalirudin, lepirudin 023 ludon or desirudin.In another exemplary embodiment, A is the pipe of the first cellulosic polymers support tube or filling.One or more fiber of the first cellulosic polymers support is aligned.In another exemplary embodiment, the first cellulosic polymers support is seamless.In another exemplary embodiment, the first cellulosic polymers support is integrally formed.
[0011] in another exemplary embodiment, described biomolecule or anti-platelet agents are covalently bound to first cellulosic polymers by junctional complex.In another exemplary embodiment, described compositions comprises that at least one has the part of following structural formula:
A wherein
1Be as defined herein.In another exemplary embodiment, described at least one part has following structural formula:
In another exemplary embodiment, described at least one part has following structural formula:
In another exemplary embodiment, hirudin is to be selected from following member: wild type hirudin, hirudin analog, bivalirudin, lepirudin 023 ludon or desirudin.In another exemplary embodiment, anti-platelet agents comprises the thrombin binding site.In another exemplary embodiment, anti-platelet agents comprises protein sequence, and it is to be selected from following member: FPRP and LYEEPIEEFDGN.In another exemplary embodiment, the first cellulosic polymers support is seamless.In another exemplary embodiment, the first cellulosic polymers support is integrally formed.
[0012] in another exemplary embodiment, described at least one part has following structural formula:
X wherein
1Be independently selected from following member with X2: covalent bond, O, S, NH or NR*, wherein R* is selected from following member: alkyl replacement or unsubstituted, that replace or unsubstituted assorted alkyl, that replace or unsubstituted cycloalkyl, Heterocyclylalkyl replacement or unsubstituted, aryl replacement or unsubstituted, that replace or unsubstituted heteroaryl, L
1Be to be selected from following member: water solublity and insoluble polymer.In another exemplary embodiment, L
1Comprise and be selected from following member: polyphosphazene, poly-(vinyl alcohol), polyamide, Merlon, polyalkylene, polyacrylamide, poly alkylene glycol, polyalkylene oxides, polyalkylene terephthalates, polyvinylether, polyvinyl ester, polyvinyl halides, polyvinylpyrrolidone, polyglycolide, polysiloxanes, polyurethane, poly-(methyl methacrylate), poly-(ethyl methacrylate), poly-(butyl methacrylate), poly-(isobutyl methacrylate), poly-(N-Hexyl methacrylate), poly-(isodecyl methacrylate), poly-(lauryl methacrylate), poly-(phenyl methacrylate), poly-(acrylic acid methyl ester .), poly-(isopropyl acrylate), poly-(Isobutyl 2-propenoate), poly-(octadecyl acrylate), polyethylene, polypropylene, poly-(ethylene glycol), poly-(oxirane), poly-(PETP), poly-(vinyl acetate), polrvinyl chloride, polystyrene, polyvinylpyrrolidone, Pluronic, polyvinyl phenol, saccharide (glucosan for example, amylose, hyaluronic acid, poly-(sialic acid), heparan, heparin, or the like); Poly-(aminoacid), for example poly-(aspartic acid) and poly-(glutamic acid); Nucleic acid and copolymer thereof.In another exemplary embodiment, L
1Comprise and be selected from following member: peptide, saccharide, polyethers, polyamine, polycarboxylic acids, poly alkylene glycol, such as Polyethylene Glycol (＂ PEG ＂), polypropylene glycol (＂ PPG ＂), copolymer of ethylene glycol and propylene glycol or the like, poly-(oxyethylation polyhydric alcohol), poly-(enol), poly-(vinylpyrrolidone), poly-(hydroxypropyl methyl acrylamide), poly-(alpha-hydroxy acid), poly-(vinyl alcohol), poly-phosphorus piperazine, poly-oxazoline, poly-(N-acryloyl morpholine), Polysialic acid, polyglutamate, polyaspartic acid salts, polylysine, polymine, biodegradable polymer (for example polyactide, polyglycerin ester and copolymer thereof), polyacrylic acid.In another exemplary embodiment, L
1Be to be selected from following member: PEG, PPG and copolymer thereof.In another exemplary embodiment, L
1Be PEG, wherein said PEG comprises many monomer subunits, and its number is the integer from about 1-about 5000.In another exemplary embodiment, described many monomer subunit numbers are integers of from about 10 to about 1000.In another exemplary embodiment, described many monomer subunit numbers are integers of from about 10 to about 500.In another exemplary embodiment, described many monomer subunit numbers are integers of from about 20 to about 400.In another exemplary embodiment, described many monomer subunit numbers are integers of from about 20 to about 250.In another exemplary embodiment, described many monomer subunit numbers are integers of from about 50 to about 200.In another exemplary embodiment, described many monomer subunit numbers are integers of from about 50 to about 125.In another exemplary embodiment, described many monomer subunit numbers are integers of from about 50 to about 100.In another exemplary embodiment, described many monomer subunit numbers are integers of from about 60 to about 90.In another exemplary embodiment, described many monomer subunit numbers are integers of from about 60 to about 90.In another exemplary embodiment, the first cellulosic polymers support is seamless.In another exemplary embodiment, the first cellulosic polymers support is integrally formed.
[0013] in another exemplary embodiment, described at least one part has following structural formula:
X wherein
1, L
1And X
2As defined herein.In another exemplary embodiment, X
2Be connected to the C-terminal of hirudin.In another exemplary embodiment, X
2Be connected to the aminoacid of the C-terminal of hirudin.In another exemplary embodiment, X
2Side chain by aspartic acid or glutamic acid part is connected to hirudin.In another exemplary embodiment, X
2Side chain by aspartic acid or glutamic acid part is connected to hirudin.In another exemplary embodiment, X
2Be connected to the amino acid moiety of hirudin, it is to be selected from following member: D5, E8, E17, D33, E35, E43, D53, D55, E57, E58, E61, E62 and Q65.In another exemplary embodiment, anti-platelet agents comprises the thrombin binding site.In another exemplary embodiment, anti-platelet agents comprises protein sequence, and it is to be selected from following member: FPRP and LYEEPIEEFDGN.In another exemplary embodiment, the first cellulosic polymers support is seamless.In another exemplary embodiment, the first cellulosic polymers support is integrally formed.
[0014] in another exemplary embodiment, described at least one part has following structural formula:
X wherein
1, L
1And X
2As defined herein.In another exemplary embodiment, L
1Be PEG.In another exemplary embodiment, hirudin connects by its end structure territory.In another exemplary embodiment, X
2Be connected to the aminoacid of the C-terminal of hirudin.In another exemplary embodiment, X
2Side chain by aspartic acid or glutamic acid part is connected to hirudin.In another exemplary embodiment, X
2Side chain by aspartic acid or glutamic acid part is connected to hirudin.In another exemplary embodiment, X
2Be connected to the amino acid moiety of hirudin, described part is to be selected from following member: D5, E8, E17, D33, E35, E43, D53, D55, E57, E58, E61, E62 and Q65.In another exemplary embodiment, anti-platelet agents comprises the thrombin binding site.In another exemplary embodiment, anti-platelet agents comprises protein sequence, and it is to be selected from following member: FPRP and LYEEPIEEFDGN.
[0015] in another exemplary embodiment, the invention provides the compositions that comprises following structural formula II I:
A---X
1---L
1---X
2---L
2---anti-platelet agents
Formula III
Wherein A is the first cellulosic polymers support, X
1Be to be selected from the first following member: covalent bond, O, S, NH or NR, L
1Be first junctional complex of choosing wantonly, X
2Be to be selected from the second following member: covalent bond, O, S, NH or NR, L2 are second junctional complexs of choosing wantonly, and anti-platelet agents is to be selected from following member, include but not limited to: heparin, hirudin, bivalirudin, lepirudin 023 ludon, desirudin, argatroban, dabigatran, the dabigatran ester, melagatran, Xi Meijia group and prodrug thereof.
[0016] in another exemplary embodiment, A is the pipe of the first cellulosic polymers support tube or filling, and wherein one or more fiber of the first cellulosic polymers support is aligned.In another exemplary embodiment, L
1With L2 is to be selected from following member independently of one another: Polyethylene Glycol and polypropylene glycol.In another exemplary embodiment, each X is N, and L
1Each is selected from following member naturally with L2: Polyethylene Glycol and polypropylene glycol.In another exemplary embodiment, anti-platelet agents is to be selected from following member: hirudin, hirudin analog, bivalirudin, lepirudin 023 ludon or desirudin.
[0017] in another exemplary embodiment, the invention provides the compositions that comprises as shown in the formula IV:
Formula IV
Wherein A is the first cellulosic polymers support, and each L is first junctional complex independently, and it is independently selected from Polyethylene Glycol and polypropylene glycol, and wherein m is from about 0 to about 100, from about 1 to about 50, and from about 1 to about 10 integer; And each anti-platelet agents is to be independently selected from heparin, hirudin, and bivalirudin, lepirudin 023 ludon, desirudin, argatroban, dabigatran, the dabigatran ester, melagatran, Xi Meijia group and prodrug thereof, wherein z is from 0 to 1 integer.
[0018] in another exemplary embodiment, anti-platelet agents is to be selected from following member: hirudin, and the hirudin analog, bivalirudin, lepirudin 023 ludon or desirudin, wherein z is 1.In another exemplary embodiment, each L is selected from following member: Polyethylene Glycol and polypropylene glycol.In another exemplary embodiment, A is the pipe of the first cellulosic polymers support tube or filling, and wherein one or more fiber of the first cellulosic polymers support is aligned.In another exemplary embodiment, anti-platelet agents is to be selected from following member: hirudin, hirudin analog, bivalirudin, lepirudin 023 ludon or desirudin.
[0019] in another exemplary embodiment, the invention provides the compositions that comprises following formula V:
Formula V
Wherein A is the first cellulosic polymers support, and each X is selected from the first following member: covalent bond, O, S, NH or NR, wherein n is from about 0 to about 100, from about 1 to about 50, from the integer of about 1-10; Each L is first junctional complex independently, and it is independently selected from Polyethylene Glycol and polypropylene glycol, and wherein m is from about 0 to about 100, from about 1 to about 50, and from about 1 to about 10 integer; Be independently selected from heparin with each anti-platelet agents, hirudin, bivalirudin, lepirudin 023 ludon, desirudin, argatroban, dabigatran, dabigatran ester, melagatran, Xi Meijia group and prodrug thereof.In another exemplary embodiment, anti-platelet agents is to be selected from following member: hirudin, and the hirudin analog, bivalirudin, lepirudin 023 ludon or desirudin, wherein z is from 0 to 1 integer.In another exemplary embodiment, X is that N and each L are selected from following member: Polyethylene Glycol and polypropylene glycol.In another exemplary embodiment, A is the pipe of the first cellulosic polymers support tube or filling, and wherein one or more fiber of the first cellulosic polymers support is aligned.In another exemplary embodiment, each anti-platelet agents is to be independently selected from following member: hirudin, hirudin analog, bivalirudin, lepirudin 023 ludon or desirudin.
[0020] in another exemplary embodiment, the invention provides the compositions that comprises following formula VI:
Formula VI
Wherein A is the first cellulosic polymers support, and X is selected from following member: O, S, and NH or NR and L are junctional complexs.In another exemplary embodiment, A is the pipe of the first cellulosic polymers support tube or filling.One or more fiber of the first cellulosic polymers support is aligned.In another exemplary embodiment, L is selected from following member: Polyethylene Glycol and polypropylene glycol.In another exemplary embodiment, X is that N and L are selected from following member: Polyethylene Glycol and polypropylene glycol.In another exemplary embodiment, C=O partly is the lactide part in the first cellulosic polymers support.In another exemplary embodiment, C=O partly is the Acetic acid, hydroxy-, bimol. cyclic ester part in the first cellulosic polymers support.In another exemplary embodiment, anti-platelet agents is to be selected from following member: hirudin, bivalirudin, lepirudin 023 ludon, desirudin, argatroban, dabigatran, dabigatran ester, melagatran, Xi Meijia group and prodrug thereof.In another exemplary embodiment, anti-platelet agents is to be selected from following member: hirudin, hirudin analog, bivalirudin, lepirudin 023 ludon or desirudin.
[0021] in another exemplary embodiment, the invention provides the compositions that comprises following formula VII:
Formula VII
Wherein A is the first cellulosic polymers support, and X is the member who is selected from NH, and L is that Polyethylene Glycol and anti-platelet agents are hirudins, hirudin analog, bivalirudin, lepirudin 023 ludon or desirudin.In another exemplary embodiment, C=O partly is the lactide part in the first cellulosic polymers support.In another exemplary embodiment, C=O partly is the Acetic acid, hydroxy-, bimol. cyclic ester part in the first cellulosic polymers support.
[0022] in another exemplary embodiment, all have first cellulosic polymers support in summary of the invention part and any compositions of describing herein, it is seamless.In another exemplary embodiment, all have first cellulosic polymers support in summary of the invention part and any compositions of describing herein, it is integrally formed.In another exemplary embodiment, all be that to be designed to be placed to end-end identical or end to side is identical in summary of the invention part and any compositions of describing herein.Coincide in order to hold-to hold, each side of graft all is placed with and makes natural artery and graft diameter be complementary.Can use interrupted or successive stitching thread that the two ends of graft and tremulous pulse are kept together.For end to side coincide, can cut graft at a certain angle and (be generally 45
0, but can be 0 to 90
0Between change), and place it in the side of natural artery.
[0023] in another exemplary embodiment, all comprise second polymer in summary of the invention part and any compositions of describing herein, it comprises and is selected from following member: PTFE and Dacron.In another exemplary embodiment, any compositions in summary of the invention part and description herein all further comprises sleeve, described sleeve holds the outer surface of the pipe of the first cellulosic polymers support tube or filling, but is not arranged in the chamber of the pipe of the first cellulosic polymers support tube or filling.In another exemplary embodiment, any compositions in summary of the invention part and description herein all comprises sleeve, described sleeve comprises polymer or subunit, and it is to be selected from following member: polyethylene terephthalate and politef.
[0024] in another exemplary embodiment, for in summary of the invention part and any compositions of describing herein, the described first cellulosic polymers support is seamless, the periphery alignment, and the fiber one of at least of the first cellulosic polymers support comprises poly-(lactide-co-glycolide) (PLGA), described hirudin is attached to the described first cellulosic polymers support by the junctional complex covalency, and described junctional complex is to be selected from following member: diaminourea poly-(ethylene glycol) and poly-(ethylene glycol).
[0025] in another exemplary embodiment, all has the length of about 1mm herein to about 50cm in summary of the invention part and any compositions of describing.In another exemplary embodiment, all has the length of about 0.5cm herein to about 10cm in summary of the invention part and any compositions of describing.In another exemplary embodiment, all has the length of about 3mm herein to about 6mm in summary of the invention part and any compositions of describing.In another exemplary embodiment, all has the internal diameter of about 0.01mm to 6mm in any compositions of summary of the invention part and description herein.In another exemplary embodiment, all has the length of about 3mm herein to about 6mm in summary of the invention part and any compositions of describing.In another exemplary embodiment, all has the length of about 4cm herein to about 8cm in summary of the invention part and any compositions of describing.In another exemplary embodiment, all be used as the parts of A/V shunting or hemodialysis access graft in any compositions of summary of the invention part and description herein.
[0026] in yet another aspect, the invention provides the method for treatment experimenter damage, described method comprises that (i) is with the amount application invention overview section that is enough to treat described damage under certain condition or compositions as herein described target site to described experimenter.In another exemplary embodiment, described target site is to be selected from following member: coronary artery, and femoral artery ， popliteal tremulous pulse, carotid artery, cerebral arteries, abdominal part, above knee, the following and radius of knee.In another exemplary embodiment, described target site is to be selected from following member: the vascular tissue that carotid artery and knee are following.In another exemplary embodiment, damage relates to the blood vessel of cut-out, the described first cellulosic polymers support has the shape of the pipe of pipe or filling, it comprises first end and second end, the blood vessel of described cut-out comprises the first blood vessel stump and the second blood vessel stump, and described application comprises: (ii) first end with described compositions is connected to the described first blood vessel stump; (iii) second end with described compositions is connected to the described second blood vessel stump.
[0027] in yet another aspect, the invention provides the method for the angiogenic growth that strengthens the experimenter, described method comprises: (i) with the amount application invention overview section that is enough to strengthen angiogenic growth under certain condition or compositions as herein described target blood site to described experimenter.
[0028] in another exemplary embodiment, this polyalkylene oxide is selected from as follows: poly(ethylene oxide), poly(propylene oxide) and combination thereof.In another exemplary embodiment, the present invention further comprises cell.In another exemplary embodiment, this cell embedding is in the first cellulosic polymers support or on the surface of the first cellulosic polymers support.In another exemplary embodiment, this cell is selected from stem cell and CFU-GM.In another exemplary embodiment, this cell is selected from as follows: the vascular cell of growing up, blood vessel CFU-GM, the blood vessel stem cell, the myocyte that grows up, muscle CFU-GM, muscle stem cell, adult neurocyte, neural progenitor cell, neural stem cell, Schwann cell, fibroblast, the Skin Cell of growing up, skin CFU-GM and skin progenitor cell.In another exemplary embodiment, the present invention further comprises direct or passes through the covalently bound molecule to the described first cellulosic polymers support of junctional complex, and described molecule can covalency or non-covalent being connected to be selected from following member: extracellular matrix component, somatomedin, differentiation factor and combination thereof.In another exemplary embodiment, this molecule is covalently bound by junctional complex, and this junctional complex is selected from as follows: two-amino poly-(ethylene glycol), and poly-(ethylene glycol) and combination thereof.In another exemplary embodiment, this molecule is selected from as follows: heparin, Heparan sulfate, heparan sulfate proteoglycan and combination thereof.In another exemplary embodiment, this extracellular matrix component is selected from as follows: laminin, collagen, fibronectin, elastin laminin, vitronectin, Fibrinogen, polylysine and combination thereof.In another exemplary embodiment, somatomedin is selected from as follows: acid fibroblast growth factor, basic fibroblast growth factor, nerve growth factor, neurotrophic factor derived from brain, insulin like growth factor, platelet derived growth factor, transforming growth factor, vascular endothelial cell growth factor, epidermal growth factor, keratinocyte growth factor and combination thereof.In another exemplary embodiment, differentiation factor is selected from as follows: stroma cell derivative factor, and Shh albumen (sonic hedgehog), bone morphogenetic protein is incised (notch) part, Wnt and combination thereof.In another exemplary embodiment, the described first cellulosic polymers support has tubulose, fill tubulose or shaft-like, and wherein said polymer is seamless.
[0029] in another exemplary embodiment, said composition is coated onto on the rotation mandrel by the polymer solution that will comprise polymer and makes.In another exemplary embodiment, described polymer support has thin slice, pipe or fill the shape of pipe and make by the electrostatic spinning process that comprises the rotation mandrel with at least one non-conductive area.In another exemplary embodiment, described polymer support has shaft-like and makes by the electrostatic spinning technology that comprises the rotation mandrel with air gap.
[0030] in another exemplary embodiment, the invention provides the pharmaceutical composition that comprises following component: (a) compositions described herein; (b) pharmaceutically acceptable excipient.In another exemplary, said composition is shaft-like or tubulose and the wherein at least a first cellulosic polymers scaffold fibers comprise poly-(lactide-copolymerization-Acetic acid, hydroxy-, bimol. cyclic ester) (PLGA).In another exemplary, said composition has the length of about 0.5cm-50cm.In another exemplary, the present invention further comprises the sleeve pipe around the first cellulosic polymers support.In another exemplary, this sleeve pipe comprises the second cellulosic polymers support, and described second cellulosic polymers is aligned or has random orientation.In another exemplary, the present invention further comprises first sleeve pipe of first end that centers on the first cellulosic polymers support and centers on second sleeve pipe of second end of the first cellulosic polymers support.
[0031] in yet another aspect, the invention provides the method for treatment experimenter damage, described method comprises: (i) with the amount that is enough to treat described damage be enough to treat the target site that under the condition of described damage compositions described herein is applied to described experimenter.In another exemplary, described damage is selected from as follows: the nerve of disconnection, the nerve of damaged, the muscle of disconnection, the muscle of damaged, the blood vessel of disconnection, the blood vessel of damaged, the skin of skin wound and contusion.In another exemplary, this damage comprises the nerve of disconnection, the described first cellulosic polymers support is the tubulose that comprises first end and second end, fill tubulose or shaft-like, and the nerve of described disconnection comprises first nerves stump and nervus opticus stump, and described using comprises: (ii) first end of described compositions is connected to described first nerves stump and (iii) described second end of described compositions is connected to described nervus opticus stump.
[0032] in another exemplary, described damage relates to the nerve of damaged, and described use comprise be selected from as follows: (ii) compositions described herein is wrapped in around the nerve of described damaged, wherein said compositions has the shape of thin slice.In another exemplary, damage comprises the nerve of damaged, and described use comprise be selected from as follows: (ii) compositions is inserted the nerve of described damaged, the wherein said first cellulosic polymers support is shaft-like, tubulose or fill tubulose.In another exemplary, the invention provides the method that strengthens experimenter's nerve growth, described method comprises: (i) with the amount that is enough to strengthen nerve growth be enough to strengthen the target site that under the condition of nerve growth compositions described herein is applied to described experimenter.In another exemplary embodiment, this damage relates to the skin of incised wound or the skin of contusion, and the described first cellulosic polymers support has the shape of thin slice, and shown in use and comprise: (i) described compositions is attached on the skin of described incised wound; Thereby treat described damage.In yet another aspect, the invention provides the method that strengthens experimenter's skin growth, the wherein said first cellulosic polymers support has the shape of thin slice, and described method comprises: (i) with the amount that is enough to strengthen skin growth be enough to strengthen under the condition of skin growth on the site that compositions as herein described is applied to the skin that described experimenter need treat.
[0033] in another exemplary, this damage comprises: the blood vessel of disconnection, the described first cellulosic polymers support is to comprise the tubulose of first end and second end or fill tubulose, and the blood vessel of described disconnection comprises the first blood vessel stump and the second blood vessel stump, and described using comprises: (ii) described first end with described compositions is connected to the described first blood vessel stump; And (iii) described second end with described compositions is connected to the described second blood vessel stump.In yet another aspect, the invention provides the method that strengthens experimenter's angiogenic growth, described method comprises: (i) with the amount that is enough to strengthen angiogenic growth with being enough to strengthen under the condition of angiogenic growth compositions described herein is applied to the vascular site that described experimenter need treat.
[0034] in another exemplary, this damage comprises the muscle of disconnection, the described first cellulosic polymers support is the tubulose that comprises first end and second end, fill tubulose or shaft-like, and the muscle of described disconnection comprises the first muscle stump and the second muscle stump, and described using comprises: (ii) described first end with described compositions is connected to the described first muscle stump; And (iii) described second end with described compositions is connected to the described second muscle stump.In another exemplary, described damage relate to damaged muscle and described use comprise be selected from as follows: (ii) compositions described herein is wrapped in around the muscle of described damaged, wherein said compositions has the shape of thin slice.In another exemplary, this damage relates to the muscle of damaged, and described use comprise be selected from as follows: compositions is inserted the muscle of described damaged, and the wherein said first cellulosic polymers support has bar, pipe or fills the shape of pipe.In yet another aspect, the invention provides the method that strengthens experimenter's muscle growth, described method comprises: (i) compositions described herein is applied to the muscle site that described experimenter need treat under the condition of muscle growth with the amount that is enough to strengthen muscle growth with being enough to strengthen.The invention provides in yet another aspect and make method for compositions described herein.In another exemplary, described method comprises: (i) one or more fiber is carried out the electrostatic spinning process, thereby make described compositions.In another exemplary, wherein said electrostatic spinning process comprises the rotation mandrel with air gap or at least one non-conductive area.
[0035] in second aspect, the invention provides the mandrel that is used for electrostatic spinning equipment, comprise: first conduction region; Second conduction region; And the non-conductive area that between first and second conduction regions, extends, wherein the non-conductive area is adjusted the cellulosic polymers that size and configuration are configured to accept to be used to form the first cellulosic polymers support.In another exemplary, described non-conductive area is to place this mandrel sleeve pipe on every side.In another exemplary, described non-conductive area is selected from as follows: adhesive tape, insulating tape, politef, and plastics.In another exemplary, described non-conductive area is interconnection with two conduction mandrel districts.In another exemplary, the discontinuous part of described non-conductive area between two conduction mandrel districts, extending.In another exemplary, described non-conductive area is selected from politef and plastics.In another exemplary, described non-conductive property have be selected from greater than with diameter less than described conduction region.
[0036] in the third aspect, the invention provides the mandrel that is used for electrostatic spinning equipment, comprise: first conduction region and second conduction region have wherein formed non-conductive area between first and second conduction regions at the air gap between first and second conduction regions.In another exemplary embodiment, the present invention further comprises: be positioned at first non-conductive sleeve at least a portion of first current-carrying part and be positioned at second non-conductive sleeve at least a portion of second current-carrying part.In another exemplary, the mandrel with non-conductive area combines with the electrostatic spinning system.In another exemplary, the mandrel with air gap combines with the electrostatic spinning system.
Description of drawings
[0037] Fig. 1 is the sketch map with electrostatic spinning equipment of mandrel 56A.
[0038] Fig. 2 is the perspective view with electrostatic spinning equipment of mandrel 56A.
[0039] Fig. 2 A is the part of the electrostatic spinning equipment with mandrel 56A of formation polymer support 90.
[0040] Fig. 2 B is the part of the electrostatic spinning equipment with mandrel 56A of formation polymer support 90.
[0041] Fig. 3 has illustrated the various mandrels that are used to make the cellulosic polymers support.(A) wherein whole surface is conduction mandrel 56; (B) has the first conduction region 57A, the second conduction region 57B, the mandrel 56A of non-conductive area 55, the first interface 55A and second contact surface 55B; (C) wherein non-conductive area 55 with the cross section of two conduction mandrel interconnected mandrel 56A in district; (D) wherein non-conductive area 55A is the sleeve pipes of covering current-carrying part 57 surface parts; (E) has the first conduction region 57A, the second conduction region 57B, the first conduction region face 57C, the mandrel 56B of the second conduction region face 57D.This conduction region is separated by air gap 58.
[0042] Fig. 4 A sketch map of the vertically aligned fibrous tubular polymer support of micrometer/nanometer of serving as reasons.The serve as reasons sketch map of the vertically aligned fibrous shaft-like polymer support of micrometer/nanometer of Fig. 4 B.Annotate: fiber size is not to draw in proportion.
[0043] Fig. 5 A is the sketch map of pipe cross section among the displayed map 4A.Fig. 5 B is the sketch map of displayed map 4B king-rod cross section.
[0044] Fig. 6 is the sketch map with electrostatic spinning equipment of mandrel 56B.
[0045] Fig. 7 is the perspective view with electrostatic spinning equipment of mandrel 56B.
[0046] Fig. 7 A is the part of the electrostatic spinning equipment with mandrel 56B of formation polymer support 92.
[0047] Fig. 8 is the sketch map of vertically aligned polymer support thin slice 96.
[0048] Fig. 9 uses the process chart that is generated the roll compacting process of the cellulosic polymers pipe holder with seam by arranged polymeric support thin slice for showing.Vertically aligned herein polymer support thin slice 96 roll compacting around bar 97, stitching or bonding then.
[0049] Figure 10 comprises the sketch map of aligned thin slice 96 and 100 " decussation " thin slice 102.
[0050] Figure 11 is the sketch map with many spinning heads electrostatic spinning equipment of mandrel 56B.This polymer solution 38,38A and 38B comprise the polymer that is dissolved in solvent, and are included in ejection assemblies 36 respectively, among 36A and the 36B.This ejection assemblies is the part of jet pump assembly 32, wherein computer 34 by controlled pressure and flow speed control polymer solution leave the speed of ejector.Randomly, can provide and control different flow velocitys selected spinning head.Flow velocity can depend on the required physical characteristic of polymer support, that is, the thickness of film, fibre diameter, aperture size, the density of film etc., and change.
[0051] this jet pump assembly 32 with feed of polymer solution to the spinning head 42 that is positioned at platform 44,42A and 42B.This spinning head is pointed, and it allows jet to form and pass through not have to hinder.Utilize high voltage power supply 48 scope to be applied on this spinning head to the voltage of about 30kV for about 10kV by electric wire 41A.
[0052] mandrel 56B (it is mentioned in Fig. 3 B, comprises 57A, 57B and 58) is positioned at spinning head 42, thereby the following of 42A and 42B forms electric field between charged spinning head and mandrel 56A.This electric field makes a branch of polymer solution from the ejection of this spinning head and be ejected into mandrel 56B, forms the filament or the fiber 46 of micron or nanometer diameter, 46A and 46B.This drill chuck is used earth lead 41B and 41C ground connection.
[0053] this mandrel 56B is connected to first drill chuck 54 (being connected to non-conductive bearing 60) and the second drill chuck 54A that links to each other with motor 52 (being connected to non-conductive bearing 60A).This motor 52 is connected with the speed control 50A of control motor rotation mandrel 56B speed.Randomly, can provide different rotary speed.Rotary speed can depend on the required physical characteristic of polymer support, that is, the thickness of film, fibre diameter, aperture size, the density of film etc., and change.
[0054] Figure 12 is the SEM image of non-aligned (A) and aligned (B) DEPLLA nanofiber.C) use the exemplary plot of the covalent bond of heparin and bFGF and laminin for having shown to the chemical modification of PLLA nanometer.Improved elisa technique is used to show that bFGF is connected the relative level of undressed two-NH2-PEG polystyrene surface (E) that applies with PLLA nanofiber (D) the heparin functionalization and poly-(acrylic acid) that modify.
[0055] Figure 13 is the neurite that extends from the tissue of the DRG on non-aligned nanofiber.The immunofluorescence dyeing of neurofilament be used for from In vitro culture after 6 days undressed, the neurite that DRG on the non-aligned nanofiber of LAM and LAM+bFGF tissue extends develops.Scale=200 μ m.
[0056] Figure 14 is the neurite that the DRG tissue from the non-aligned nanofiber extends.The immunofluorescence dyeing of neurofilament be used for In vitro culture after 6 days undressed, the neurite that the DRG tissue on the aligned nanofiber of LAM and LAM+bFGF extends develops.Nanofiber is arranged with vertical direction.Scale=200 μ m.
[0057] Figure 15 is that the height of the neurite form on non-aligned and aligned LAM+bFGF PLLA nanofiber amplifies the confocal microscopy image.Nanofiber is aligned with vertical direction.
[0058] Figure 16 cultivates human mescenchymal stem cell for coccoid by 1,3 on PLLA micrometer/nanometer fibrous membrane, or 6 days.Cell demonstrates along with the time moves and random alignment gradually at non-aligned micrometer/nanometer fiber.Show during at 3 and 6 days in the enhanced migration of machine direction and along the overall alignment of machine direction at the cell on the aligned micrometer/nanometer fiber.Scale=200 μ m.
[0059] Figure 17 is the wound healing models of various cell types on the micrometer/nanometer fibrous framework.MSCs: mescenchymal stem cell two days later.Foreskin fibroblast after FFs:5 days.ECs: the endotheliocyte after a day.On non-aligned micrometer/nanometer fibrous framework, the coverage of wound from minimum (MSC sample) to medium (EC sample).When micrometer/nanometer cell and wound major axis (A-Para) when being arranged in parallel, move to the cell major injury in the wound.When the micrometer/nanometer fiber is arranged perpendicular to the wound major axis, cell migration and wound covering degree maximum.
[0060] Figure 18 has proved at A for the fluorescence staining to actin (phalloidin) and nuclear (iodate third ingot)) common carotid artery and B in the natural body) cell skeletal structure between the arrangement nanofiber polymer flake of the smooth muscle cells inoculation of choosing is similar.
[0061] Figure 19 is the structure of blood vessel graft of the embedding stem cell of nanofiber.A) stem cell is inoculated on the aligning flakes of biodegradable nanofiber.B) tubular structure generates by roll compacting thin slice around bar.C) remove the shape that graft is kept in bar and use stitching.
[0062] Figure 20 is the Verhoeff dyeing of blood vessel graft and rat artery cross section.1 to 3 week that was grown in of collagen (redness) and elastin laminin (black) fiber significantly improves.Till three weeks, tissue and the natural rat artery of handling blood vessel graft have strong similar figures.
[0063] Figure 21 is for implanting the immunohistochemical staining (brown) of three week back CD31 (endothelial cell marker) cross sections in blood vessel graft.A) rat artery.B) blood vessel graft after three weeks.
[0064] Figure 22 is at the immunohistochemical staining (brown) of implanting 3 week back α-Ji Dongdanbai (smooth muscle labelling) cross sections in blood vessel graft.A) rat artery.B) blood vessel graft after three weeks.
[0065] Figure 23 is for when sarcoplast grows on non-aligned and non-patterned surface, and myotube forms with random fashion, and when sarcoplast grew on arrangement nanofiber and micropatterned surface, this myotube formed in aligned mode.
[0066] Figure 24: the set of myoblastic arrangement and myotube on arrangement PLLA nano fiber scaffold.The SEM image has shown that (A) is orientated and (B) structure of aligned nano fiber scaffold immediately, then in differentiation culture liquid after 3 days sarcoplast at (C) random orientation and (D) F-actin immunofluorescence dyeing on the aligned nano fiber scaffold.The immunofluorescence dyeing that carries out skeleton MHC be presented at random (E, G) and arrange (F, H) nano fiber scaffold 3 days (E, F) and 7 days (G, myotubes H).At (I) random orientation and (J) arrange the base material painted low amplification of skeleton MHC overall arrangement and length of myotube that merged pictorial display after last 7 day.Arrow is represented the direction of nanofiber.The arrow of E and F is represented nuclear.Scale be respectively 50 μ m (A-H) and 100 μ m (I, J).
[0067] Figure 25 is the quantification of myotube tissue and form on aligned fiber base material.(A) at the arrangement angle of nanofiber direction myotube.(B) length of myotube after 7 days.(C) width of myotube after 7 days.* shown statistically evident difference (P＜0.05).
[0068] Figure 26 is the quantification that sarcoplast breeds and the myotube striped forms on aligned fiber base material.(A) BrdU of cell proliferation mixes (R, Ran; A, Align).(B) be presented at the immunofluorescence dyeing (scale: 20 μ m) of arranging the anti--MHC of striated myotube on the nano fiber scaffold.(C) quantification of striated cell percentage ratio after 7 days.* shown statistically evident difference (P＜0.05).
[0069] Figure 27 is that sarcoplast is arranged and the quantification of myotube tissue on micro-patterning PDMS base material.By (A) SEM (side view) and (B) phase contrast microscopy show micro-patterning polydimethylsiloxane substrate.F-actin distribution after 2 days in differentiation culture liquid is presented at (C) non-patterning and (D) on the micro-patterning base material.The fluorescence staining of carrying out skeleton MHC be presented at pattern-free (E, G) and micro-patterning (F, H) on the film 2 days (E, F) and 7 days (G, cell fusion H).Arrow is represented the direction of microgroove.Scale is respectively 5 μ m (A), 20 μ m (B) and 50 μ m (C-H).
[0070] Figure 28 is the quantification of sarcoplast tissue and form on the micro-patterning film.(A) on pattern-free (Con) and micro-patterning (Pat) film at the aligned angle of microgroove direction myotube.(B) width of length (C) myotube after 7 days of myotube after 7 days.* shown statistically evident difference (P＜0.05).
[0071] Figure 29 is the quantification that sarcoplast breeds and striped forms on micro-patterning PDMS film.(A) on pattern-free (Con) and micro-patterning (Pat) film, merge in early days mixing because of the BrdU of cell proliferation.(B) quantification of striated cell percentage ratio after 7 days.* shown statistically evident difference (P＜0.05).
[0072] Figure 30 scanning electron microscope image that myotube forms on nano fiber scaffold.After 7 days at A) random orientation and (B) arrangement (the scale bar: 50 μ m) of the myotube in the differentiation culture liquid on the aligned PLLA nano fiber scaffold.
[0073] Figure 31 myoblastic arrangement on the biodegradable PLGC substrate of micro-patterning.(A) scanning electron microscope image of the visual patterning microgroove that declines on the PLGC base material.(B-C) after 5 days at pattern-free B) and micro-patterning (C) PLGC substrate on myoblastic F-actin dyeing in the differentiation culture liquid.Attention: on micro-patterning PLGC base material, arrange and the nervous fiber of highly organized actin.Scale is respectively 10 μ m (A) and 50 μ m (B-C).
[0074] Figure 32 has shown the process chart that is used to generate 3 D stereo muscle fiber inoculation tubular bracket.Sarcoplast is divided into aligned myotube on aligned nano fibrous membrane.Break up after 7 days, thin slice roll compacting is become to have the tubular bracket and the sutured of bar stem.
[0075] Figure 33 describes three-dimensional tubular nanometer fibrous framework and is organized in hematoxylin and eosin (H﹠amp under low (left side) and high (right side) amplification; E) dyeing.
[0076] Figure 34 is the confocal laser microscopic image that is described in the morphocytology of interior sarcoplast of the three-dimensional tubular nanometer fibrous framework in cross section (A) and major axis (B) aspect and myotube.This sample carries out immunofluorescence dyeing to F-actin (green) and nuclear (redness).
[0077] Figure 35 is the diagram of wound healed model outside arrangement or not aligned fiber upper body.(A) using parallel or vertical non-aligned fiber with the long porch of wound or aligned fibrogenic micrometer/nanometer fiber is mesh.((B) graduation 18 gauge ejector syringe needles are positioned on this nanofiber mesh to stop cell adhesion.(C) pair cell is inoculated on the nanofiber mesh.(D) after cell adhesion is to this nanofiber, remove this pin to allow cell migration to the wound.
[0078] Figure 36 is at aligned vs.To the external wound healing model on the non-aligned nanofiber with NHDF.After 48 hours, the migration and the wound covering degree of NHDF (A) the demonstration place moderate on non-aligned micrometer/nanometer fiber, and cell is at random arranged.When fiber when arranging with the direction of the edge-perpendicular of wound (B), migration and wound covering degree improve greatly, and cell and fiber alignment are embarked on journey.When the edge of fiber and wound (C) was arranged in parallel, the wound covering degree reduced widely.Dyeing is whole actin (green) and He Xisite nuclear stain (blueness).The white point line is illustrated in 0 o'clock original edge of wound.Scale is 300 μ m.
[0079] Figure 37 is at the external wound healing model that has or do not have to have on the aligned micrometer/nanometer fiber of chemical modification NHDF.In all groups, the micrometer/nanometer fiber is perpendicular to the long edge orientation of wound.After A24 hour, demonstrate the migration and the wound covering degree of raising having NHDF on the fiber of other chemical modifications.On undressed fiber, cell not exclusively covers wound area.When laminin being added on this fiber, cell migration is faster.Further strengthen migration with solvable shape or the interpolation that is fixed to the form bFGF on the micrometer/nanometer fiber.Dyeing is whole actin (green) and nuclear (blueness).The white point line is illustrated in 0 o'clock original edge of wound.The S scale is 300 μ m.
[0080] Figure 38 is the assembling of multilamellar micrometer/nanometer fibrous tissue graft.Each micrometer/nanometer sheets of fibres has the structure of labyrinth with generation at the top of each other lamination.This figure has described the assembling with decussation fibre structure graft.The fibre orientation of responsible each the individual thin slice of other structures generates.
[0081] Figure 39 is the manufacturing of micro-patterning polymeric film.(A) negative photoresist rotary coating and be exposed to ultraviolet on the silicones wafer by photomask.(B) development of the photoresist of no polymerizable ultraviolet is come, and stays patterned surface.(C) this polymer solution is cast on the wafer, spin coated, and allow polymerization.(D) then this thin film is peeled off from silicon wafer.
[0082] Figure 40 many cells type graft.(A) the nanofiber thin slice of arrangement or random orientation.(B) the inoculation many cells type on the thin slice zones of different.(C) diverse location at graft has generated the tubular structure with many cells type.
[0083] Figure 41 has illustrated that the present invention has the electrostatic spinning equipment of rotary drum colelctor electrode.
[0084] Figure 42 shows the cross section of blood vessel graft and rat artery.The blood vessel graft of implanting comprises PLLA, and does not comprise the banded biomolecule such as hirudin of covalency.Figure 42 A shows and stands painted blood vessel graft of Verhoeff ' s and rat artery.Figure 42 B shows blood vessel graft and the rat artery by immunohistochemical staining (brown) with CD31 (endothelial cell marker thing).Figure 42 C shows blood vessel graft and the rat artery that carries out immunohistochemical staining (brown) with α-Ji Dongdanbai (smooth muscle cell label).Figure 42 D shows blood vessel graft and the rat artery that carries out immunohistochemical staining with CD68.
[0085] Figure 43 shows the cross section of blood vessel graft and rat artery.The blood vessel graft of implanting comprises the banded PLLA of covalency.Figure 43 A shows and stands painted blood vessel graft of Verhoeff ' s and rat artery.Figure 43 B shows with CD31 (endothelial cell marker thing) by immunohistochemical staining (brown) blood vessel graft and rat artery.Figure 43 C shows blood vessel graft and the rat artery that carries out immunohistochemical staining (brown) with α-Ji Dongdanbai (smooth muscle cell label).Figure 43 D shows blood vessel graft and the rat artery that carries out immunohistochemical staining with CD68.
DESCRIPTION OF THE PREFERRED
Definition and abbreviation
[0086] abbreviation used herein has implication common in the chemistry and biology field usually.
[0087] singulative used herein "/should " comprises that plural number refers to, unless context has difference to indicate in addition.
[0088] used herein, except as otherwise noted, the meaning that compositions " does not contain " a kind of component substantially is that compositions contains less than about 20wt%, for example less than about 10wt%, and less than about 5wt%, or less than this component of about 3wt%.
[0089] ＂ peptide ＂ is meant that monomer wherein is aminoacid and the polymer that combines by amido link, perhaps is called as polypeptide.In addition, also comprise alpha-non-natural amino acid, for example, Beta-alanine, phenylglycine and homoarginine.The non-genomic amino acids coding also can be used for the present invention.In addition, modified comprising reactive group, glycosylation site, polymer, the treatment part, the aminoacid of biomolecule etc. also can be used for the present invention.All used aminoacid of the present invention are D-or L-isomer.In addition, other plan peptides also are useful in the present invention." peptide " used herein is meant glycosylated and nonglycosylated peptide.Also comprise the incomplete glycosylated peptide of system by expressing described peptide.Summary please refer to, Spatola, A.F., inChemistry and Biochemistry of aminoacid, peptide s and Proteins, B.Weinstein，eds.，Marcel?Dekker，New?York，p。267(1983)。
[0090] term " aminoacid " is meant natural existence and synthetic aminoacid, and the amino acid analogue and the amino acid analog thing that act in a similar manner with naturally occurring aminoacid.Naturally occurring aminoacid is those aminoacid by genetic code coding, and those aminoacid of modified, for example hydroxyproline, Gla and O-phosphoserine afterwards.Amino acid analogue is meant the chemical compound that has identical basic chemical structure with naturally occurring aminoacid, that is, with hydrogen, carboxyl, the bonded α carbon of amino and R base, homoserine for example, nor-leucine, methionine sulfoxide, methionine methyl sulfonium.These analog have the R base (for example, nor-leucine) of modification or the peptide main chain of modifying, but have kept the basic chemical structure identical with naturally occurring aminoacid.The amino acid analog thing is meant the chemical compound that has with the common chemical constitution different structure of aminoacid, but acts in a similar fashion with naturally occurring aminoacid.
[0091] " nucleic acid " used herein is meant DNA, RNA, strand, two strands, or higher accumulative hybridization motif and its any chemical modification.Modification includes, but not limited to provide the introducing additional charge, polarizability, and hydrogen bonding, electrostatic interaction, junction point and degree of functionality are to the chemical group of nucleic acid ligands base or nucleic acid ligands integral body.These modifications include, but not limited to peptide nucleic acid(PNA) (PNA), and phosphodiester group (is for example modified, thiophosphate, methyl-phosphonate), 2 '-position is sugar-modified, and 5-position pyrimidine is modified, 8-position purine is revised, the modification of the outer amine of ring, the replacement of 4-thiourea glycosides, the replacement of 5-bromine or 5-iodouracil; Backbone modifications, methylation, the rare bases pairing is in conjunction with for example different base, different cytidine and isoguanine etc.Nucleic acid can also comprise non-natural base, for example, and nitroindoline.Modification can also comprise 3 ' and 5 ' modify and for example use fluorophor (for example, quantum dot) or another part to add medicated cap.
[0092] " antibody " used herein typically refers to specific bond and the antigenic polypeptide that comprises from the framework region of immunoglobulin or its fragment or immunoconjugates of identification.The immunoglobulin of generally acknowledging comprises κ, λ, alpha, gamma, δ, ε and μ constant region gene, and countless immune globulin variable region gene.Light chain is categorized as κ or λ.Heavy chain is categorized as γ, μ, and α, δ, or ε, it defines immunoglobulin class, IgG, IgM, IgA, IgD and IgE successively respectively.
[0093] polymer that contains a more than kind type subunit described in term used herein " copolymer ".This term comprises and comprises 2,3,4,5, or the polymer of 6 types of subunits.
[0094] term " isolating " is meant that material is basically or in essence free from the component that is used for making this material.Said composition purity range following be limited to about 60%, about 70% or about 80% and purity range on be limited to about 70%, about 80%, about 90% or greater than about 90%.
[0095] " hydrogel " is meant water-fast and cross linked polymer water-swellable, and this polymer can absorb at least 3 times, the liquid of preferred at least 10 times of himself weight." hydrogel " and " temperature-sensitive polymer " is used interchangeably at this paper.
[0096] term used herein " connection " comprises and includes, but not limited to covalent bonding, ionic bonding, chemisorbed, the interaction of physical absorption and combination thereof.
[0097] term " biomolecule " or " biological organic molecule " are meant the organic molecule of being made by living body biological usually.This comprises, for example, comprises nucleotide, aminoacid, sugar, fatty acid, steroidal, nucleic acid, polypeptide, peptide, fragments of peptides, carbohydrate, lipid, and the molecule (for example, glycoprotein, ribonucleoprotein, lipoprotein etc.) of combination.
[0098] " micromolecule " is meant molecular weight less than 1kD, preferably less than the molecule of 600D.
[0099] ＂ present composition ＂ used herein is meant compositions discussed in this article, these compositions pharmaceutically acceptable salt and prodrugs.
[0100] when writing by conventional chemical formula appointment substituent group from left to right, they comprise the substituent group that chemically is equal to equally, and it can obtain by writing this structure from right to left, for example, and-CH
2O-also is intended to be expressed as-OCH
2
[0101] medicine of " effectively " amount, preparation, or penetrating agent is meant that the amount of activating agent is enough to provide the effect of required part or whole body." effectively local ", " effective in the beauty treatment ", " pharmaceutically effective " or " treatment is effectively " is the amount that has guided the required medicine of required therapeutic outcome.
[0102] " " be meant the salt that comprises The compounds of this invention, it prepares with nontoxic relatively acid or alkali (depending on the specified substituent of finding on the chemical compound described herein) pharmaceutically acceptable salt term.When chemical compound of the present invention contained relatively tart functional group, base addition salts can be by solvent-free or make the neutral form of these chemical compounds contact acquisition with the required alkali of q.s in suitable atent solvent.The example of pharmaceutically acceptable base addition salts comprises sodium salt, potassium salt, calcium salt, ammonium salt, organic amine, or magnesium salt, or similar salt.When chemical compound of the present invention contained relatively the functional group of alkalescence, acid-addition salts can be by solvent-free or make the neutral form of these chemical compounds contact acquisition with the required acid of q.s in suitable atent solvent.The example of pharmaceutically-acceptable acid addition comprises the salt derived from mineral acid, for example: hydrochlorate, hydrobromate, nitrate, carbonate, bicarbonate, phosphate, hydrophosphate, dihydric phosphate, sulfate, disulfate, hydriodate, or phosphite etc., and derived from nontoxic relatively organic acid salt, acetate for example, propionate, isobutyrate, maleate, malonate, benzoate, succinate, bolt hydrochlorate, fumarate, lactate, mandelate, phthalate, benzene sulfonate, tosilate, citrate, tartrate, mesylate etc.Also comprise amino acid whose salt, for example arginine salt etc., and acylate, for example, glucuronate or galacturonic acid hydrochlorate etc. (referring to, Berge et al for example.＂Pharmaceutical?Salts＂，Journal?of?Pharmaceutical?Science?66：1-19(1977))。Some specific compound of the present invention is double to contain alkalescence and thereby acidic functionality can make this chemical compound be converted into alkali or acid-addition salts.
[0103] neutral form of this chemical compound is preferably regenerated by making salt and alkali or acid contact and separating with parent compound in the mode of routine.The parent form of this chemical compound is at the form that is different from various salt aspect some physical property, for example dissolubility in polar solvent.
[0104] except the form of salt, the invention provides the chemical compound of prodrug forms, the prodrug of chemical compound as herein described or mixture issues biochemical variation so that chemical compound of the present invention to be provided at physiological condition easily.In addition, prodrug can be converted into chemical compound of the present invention by chemistry or biochemical process at stripped environment.
[0105] chemical compound of the present invention is also constituting the element isotope that comprises on one or more atoms of these chemical compounds with the non-natural ratio, for example, this chemical compound can by radiosiotope (for example tritium (
3H), iodine-125 (
124I) or carbon-14 (
14C)) carry out radioactive label.The isotope of all The compounds of this invention changes, and no matter is radioactive or inactive, all is intended within the scope of the present invention.
[0106] term " pharmaceutically acceptable carrier " or " pharmaceutically acceptable carrier " are meant any preparation or the mounting medium that the activating agent of the effective dose that provides defined herein is suitably sent, the bioactive effectiveness of interferon activity agent and its are enough not nontoxic to host or patient.Representational carrier comprises water, oils (vegetable oil and mineral oil), cream substrate, lotion base, ointment base etc.These substrate comprise suspending agent, thickening agent, penetration enhancers etc.These preparations are known to the professional of cosmetics and local drug world.Other information about carrier can find in following list of references: Remington:Science and Practiceof Pharmacy, 21st Ed., Lippincott, Williams ﹠amp; Wilkins (2005), it incorporates this paper by reference into.
[0107] " pharmaceutically acceptable topical carrier " and equivalent terms are meant pharmaceutically acceptable as mentioned above carrier, and it is suitable for local application.Can suspend or the lytic activity agent, and ought be administered to skin, fingernail, hair, the nonactive liquid or the paste excipient that have nontoxic and non-inflammatory character when claw or hoof are the example of pharmaceutically acceptable topical carrier.This term is specific to be intended to comprise the support material that approval is used for topical cosmetic.
[0108] term " pharmaceutically acceptable additive " is meant that field of pharmaceutical preparations is known or use and exceeds the bioactive effectiveness of interferon activity agent, and to host or the enough nontoxic antiseptic of patient, antioxidant, spice, emulsifying agent, dyestuff and excipient.The additive that is used for topical formulations is being known in the art, and can join in the topical composition, as long as they are pharmaceutically acceptable and harmless to epithelial cell or its function.In addition, they should be unable to cause the deterioration of composition stable.Part or dermal delivery preparation for example, inert filler, counter-stimulus, viscosifier, excipient, spice, opacifier, antioxidant, gellant, stabilizing agent, surfactant, softening agent, coloring agent, antiseptic, buffer agent, other penetration enhancers, and other conventional components are well known in the art.
[0109] ＂ used herein uses/administration " be meant oral administration to the experimenter, with the suppository administration, local contact, intravenous, intraperitoneal, intramuscular, in sick the damage, intranasal or subcutaneous administration, or delayed release device (for example, little osmotic pumps) is implanted.
[0110] common known carrier, diluent and/or the vehicle that is used to prepare to required purposes drug composition effective that is meant of term " excipient ".
[0111] term used herein " autogenous cell " is meant experimenter's self cell or its clone.
[0112] term used herein " homogeneous variant cell " is meant the cell that is not first experimenter self, or its clone, but is derived from second experimenter's cell or its clone, and this second experimenter is identical species with first experimenter.
[0113] term used herein " allos cell " is meant the cell that is not from first experimenter self cell, or its clone, but be derived from second experimenter's cell or its clone cell, and this second experimenter is not identical species with first experimenter.
[0114] term used herein " stem cell " is meant the cell that can be divided into other cell types, comprises that those have the cell of specific, specialization function (for example, terminally differentiated cells, erythrocyte for example, macrophage etc.).Stem cell can define (growing up/the soma cell embryonic stem cell) according to its source, or according to its potential definition (totipotency stem cell, versatility (pluripotent) stem cell, versatility (multipotent) stem cell and unipotency stem cell).
[0115] term used herein " unipotency stem cell " is meant the cell that can only produce a kind of cell type, still has the character with non-other self renewal of stem cell phase region.
[0116] " versatility (multipotent) stem cell " used herein or " CFU-GM " are meant any cell that can produce several different terminally differentiated cells types.These dissimilar cells be closely related usually (for example, hemocyte, erythrocyte for example, leukocyte and platelet).For example, mescenchymal stem cell (being also referred to as marrow stromal cell) is a multipotential cell, and can be formed into osteocyte, chondrocyte, myocyte, adipose cell, neuronal cell, and β-islet cells.Another example is the skeleton sarcoplast, and it is preferably by relating to the atomization generation Skeletal Muscle Cell that individual cells is fused to the multinuclear myotube.
[0117] term used herein " versatility (pluripotent) stem cell " is meant the cell that produces some perhaps many (yet being not whole) organism cell types.Pluripotent stem cell can be divided into any cell type in the ripe organism health, though they can not be divided into it again and derive and the cell that comes without reprogrammed.Just as is understood, " versatility (multipotent) stem cell "/CFU-GM (for example, neural stem cell) has than the narrower differentiation potential of versatility (pluripotent) stem cell.Than the more primary another kind of cell of versatility (pluripotent) stem cell (for example, must not carry out specific differentiation) is so-called " totipotency " stem cell.
[0118] term used herein " totipotency stem cell " is meant fertilized oocyte, and the cell (for example, being in the embryo of two and four cell stage of growth) that is produced by fertilized egg cell's initial division several times.Totipotent cell has the ability of the cell of any kind that can be divided into specific species.For example, single totipotency stem cell can produce whole animal body, and any in findable countless cell types in the specific species (for example, people).In this manual, versatility (pluripotent) and totipotent cell, and have the cell that is divided into complete organ or tissue potential, be called as the original ＂ stem cell of ＂.
[0119] " to dedifferente " be that phalangeal cell is returned to the more not state of specialization to term used herein.After dedifferenting, this cell will have to be divided into than possible cell type before the reprogrammed and more many or the ability of different cell type.Oppositely process of differentiation (promptly dedifferenting) may be more complicated than differentiation, and need " reprogrammed " this cell to become more original.The example that dedifferentes is that granulation promoting CFU-GM (for example early stage elementary sarcoplast) is converted into muscle stem cell or satellite cell.
[0120] " normally " stem cell is meant and does not represent unusual Phenotype or have the aberrant gene type, therefore can produce the stem cell (or its offspring) of a whole set of cell that derives from these stem cell.For example, if normal totipotency stem cell, this cell can produce, and is for example complete, the normal healthy animal.On the contrary, " unusual " stem cell for example is meant because one or more variations or genetic modification or pathogen and not normal stem cell.Therefore, unusual stem cell is different from normal stem cell.
[0121] " growing environment " is the environment of stem cell in-vitro multiplication.The feature of environment comprises the culture medium of cultured cell, and if have the supporting structure substrate of the surface of solids (for example).
[0122] " somatomedin " is meant promoting the cell growth effectively, unless join in the culture medium as a supplement, otherwise be not the material of minimal medium component.Other says it, somatomedin for not by just at the excretory molecule of cultured cells (comprise any feeder cells, if exist), even if or by in the culture medium by emiocytosis, neither enough realize adding the result's that somatomedin obtains amount by external source.Somatomedin comprises, but be not limited to, basic fibroblast growth factor (bFGF), acid fibroblast growth factor (aFGF), epidermal growth factor (EGF), insulin like growth factor-1 (IGF-I), insulin like growth factor-1 I (IGF-II), platelet derived growth factor-AB (PDGF), vascular endothelial cell growth factor (VEGF), nandrolone phenylpropionate-A, bone morphogenetic protein (BMPs), insulin, cytokine, chemotactic factor, morphogen, neutralizing antibody, other protein and micromolecule.
[0123] term used herein " differentiation factor " is meant that induced dry-cell or CFU-GM are converted into the molecule of the cell type of specific specialization.
[0124] one or more materials that the essentially identical condition of condition that provides with the synthetic extracellular matrix of feeder cells is provided for the sustenticular cell growth are provided " extracellular matrix " or " substrate ".This substrate can be provided on base material.Perhaps, a kind of component or the various ingredients that constitutes this substrate can the solution form provide.The component of extracellular matrix can comprise laminin, collagen and fibronectin.
[0125] term used herein " regeneration capacity " is meant that stem cell changes into splitted CFU-GM and the specific cell of differentiated tissues.
[0126] term used herein " self renewal " is meant that it is specified propagation that nothing is planted.
[0127] term used herein " aligned (aligned) " is meant the fibre orientation in the cellulosic polymers support, and wherein at least 50% fiber is got general direction, and its orientation has formed all axles of an arrangement.The orientation of any given fiber can depart from arranges all axles, and departs from and can represent with the described angle that forms between axle and the fibre orientation of arranging.0 ° drift angle shows accurately aligned, and 90 ° (or-90 °) show this fiber and arrange all axle arranged verticals.In an exemplary, fiber is optional from following angle with the standard deviation of arranging equal axle: 0 °-1 °, and 0 °-3 °, 0 °-5 °, 0 °-10 °, 0 °-15 °, 0 °-20 °, or 0 °-30 °.
[0128] term used herein " bar " is meant the cellulosic polymers support that is essentially solid cylindrical shape.Space and passage can be present between the single fiber that constitutes this bar.
[0129] term used herein " pipe " is meant and is essentially columned object.This pipe has inner and outer wall, internal diameter, external diameter and by the internal diameter of pipe with and the inner space determined of length.Space and passage can be present between the single fiber that constitutes this pipe.
[0130] term used herein " filling pipe " is meant the pipe that the part of this inner space is made of packing material.This packing material can be the cellulosic polymers support.Space and passage can be present between the single fiber that constitutes this pipe.
[0131] term used herein " seam " or " stitching " are meant two parts by match, connect, or the abutment that is joined together to form of overlap joint.These two parts can for example be sewed up by mechanical system, or by chemical method, for example annealing or binding agent keep together.For example, seam is by a slice region is combined formation with another zone.
[0132] term used herein " seamless " is meant and does not have seam.
[0133] term " cell " can refer to the situation of individual cells or a plurality of cells.
[0134] term used herein " extracellular matrix component " is meant and is selected from following component: laminin, collagen, fibronectin and elastin laminin.
[0135] term used herein " bracing frame " is the pipe of being made by metal except that other materials and organic polymer.When this bracing frame was made up of organic polymer, this polymer was not nanofiber as herein described or micrometer fibers polymer support.In other words, if support frame as described above is made up of the cellulosic polymers support, the average diameter of this fiber will be between 100 microns to about 50 centimetres.In some cases, whole bracing frame can be from first diameter expansion to second diameter, and wherein second diameter is greater than first diameter.
[0136] term used herein " hirudin " is meant 65 aminoacid wild type peptide or its analog.65 aminoacid wild type peptide have Folkers etc. at Biochemistry, and 28 (6); The sequence of describing among the 2601-2617 (1989).The analog of hirudin comprises peptide, a few amino acids with one or more sudden changes, and several amino acids is to the chemical modification object and the combination thereof of one or more amino acid residues.The example of hirudin comprise wild type hirudin, bivalirudin, lepirudin 023 ludon, end modified (promptly acetylizad) hirudin of end modified (promptly acetylizad) hirudin, the C-of fixed, the unvulcanised Tyr-63 hirudin in West Road, N-, the hirudin fragment (about 1-53 residue) of N-end structure territory disappearance, hirudin fragment (about 54-65 residue), [Tyr (SO that C-end structure territory lacks
3H)-63]-hirudin fragment 54-65, [Tyr (SO
3H)-63]-hirudin fragment 55-65, acetyl [Tyr (SO
3H)-63]-hirudin fragment 54-65, acetyl [Tyr (SO
3H)-63]-hirudin fragment 55-65.Being used for hirudin of the present invention can produce from multiple source.In some cases, hirudin is isolating from hirudiniasis.In other cases, the hirudin generation of from antibacterial, yeast or fungus, recombinating.Also have other situation, hirudin is chemosynthesis.The hirudin of reorganization and chemosynthesis is tending towards producing uniform product.And isolating hirudin can comprise more than one hirudin analog from hirudiniasis.Hirudin can be from (St.Louis, MO) company of a class buys such as Sigma-Aldrich.
[0137] symbol
No matter be the display part that also is perpendicular to key as key, represent that all shown part is connected in molecule, for example the point of the nubbin of polymer.
II. compositions
[0138] these compositionss can comprise polymer support.These polymer supports can be the cellulosic polymers support, for example microfibre polymer support or nanofiber polymer support.These polymer supports can also be the micro-patterning polymer supports.Compositions of the present invention and/or polymer support can be randomly for non-aligned or they for example can be vertically or the circumferential arrangement alignment.Compositions of the present invention and/or polymer support can randomly form, for example, and thin slice, decussation thin slice, pipe, the shape of bar or filling pipe.Compositions of the present invention and/or polymer support can have seam or they can be seamless.Compositions of the present invention or polymer also can randomly comprise raw material, for example: cell, biomolecule, or pharmaceutically acceptable excipient.These arrangements, shape and additional component can help to improve or regeneration or replacement biological function.The present composition does not comprise bracing frame.Said composition can be used to organizational project to improve regeneration or replacement biological function.
IIa) cellulosic polymers support
[0139] first aspect the invention provides the compositions that comprises the cellulosic polymers support.The cellulosic polymers support comprises one or more fibers that can have the certain limit diameter.In an exemplary embodiment, the average diameter of fiber is that about 0.1 nanometer is to about 50000 nanometers in the cellulosic polymers support.In another exemplary embodiment, the average diameter of fiber is that about 25 nanometers are to about 25,000 nanometers in the cellulosic polymers support.In an exemplary embodiment, the average diameter of fiber is that about 50 nanometers are to about 20,000 nanometers in the cellulosic polymers support.In an exemplary embodiment, the average diameter of fiber is that about 100 nanometers are to about 5,000 nanometers in the cellulosic polymers support.In an exemplary embodiment, the average diameter of fiber is that about 1,000 nanometer is to about 20,000 nanometers in the cellulosic polymers support.In an exemplary embodiment, the average diameter of fiber is that about 10 nanometers are to about 1,000 nanometer in the cellulosic polymers support.In an exemplary embodiment, the average diameter of fiber is that about 2,000 nanometers are to about 10,000 nanometers in the cellulosic polymers support.In an exemplary embodiment, the average diameter of fiber is that about 0.5 nanometer is to about 100 nanometers in the cellulosic polymers support.In an exemplary embodiment, the average diameter of fiber is that about 0.5 nanometer is to about 50 nanometers in the cellulosic polymers support.In an exemplary embodiment, the average diameter of fiber is that about 1 nanometer is to about 35 nanometers in the cellulosic polymers support.In an exemplary embodiment, the average diameter of fiber is that about 2 nanometers are to about 25 nanometers in the cellulosic polymers support.In an exemplary embodiment, the average diameter of fiber is that about 90 nanometers are to about 1,000 nanometer in the cellulosic polymers support.In an exemplary embodiment, the average diameter of fiber is that about 500 nanometers are to about 1,000 nanometer in the cellulosic polymers support.
[0140] in an exemplary, the cellulosic polymers support is selected from nanofiber polymer support and micrometer fibers polymer.The micrometer fibers polymer support has micro-meter scale feature (fiber diameter about 1,000 nanometer is to about 50,000 nanometer, especially about 1,000 nanometer is to about 20,000 nanometers), (about 10 nanometers of fiber diameter are to about 1 and the nanofiber polymer support has submicroscale features, 000 nanometer and especially 50 nanometers to about 1,000 nanometer).The physical arrangement of each imitated area for treatment of these polymer supports.For example natural collagen fibre or other extracellular matrix.
[0141] multiple polymers (synthetic and/or natural origin) can be used for constituting these cellulosic polymers supports.Fiber can be made by monomer or subunit.For example, can utilize lactic acid or poly-(lactic acid) or hydroxyacetic acid or polyglycolic acid to form (PLLA) (PGA) nanofiber of nanofiber or poly-(Acetic acid, hydroxy-, bimol. cyclic ester) of polylactide (PLA) or poly-(L-lactide).Thereby fiber also can form copolymer, terpolymer etc. by making more than a kind of monomer or subunit.For example, lactic acid or poly-(lactic acid) combine with hydroxyacetic acid or polyglycolic acid and form this copolymer poly-(lactide-copolymerization-Acetic acid, hydroxy-, bimol. cyclic ester) (PLGA).Be used for other copolymers of the present invention and comprise poly-(ethylene-copolymerization-vinyl alcohol).In an exemplary embodiment, fiber comprises and is selected from following polymer or subunit: aliphatic polyester, polyalkylene oxide, polydimethylsiloxane, polyvinyl alcohol, polylysine, collagen, laminin, fibronectin, elastin laminin, alginate, fibrin, hyaluronic acid, mucin, polypeptide and combination thereof.In another exemplary embodiment, fiber comprises two kinds of different polymer or the subunit that is selected from following component: aliphatic polyester, polyalkylene oxide, polydimethylsiloxane, polyvinyl alcohol, polylysine, collagen, laminin, fibronectin, elastin laminin, alginate, fibrin, hyaluronic acid, mucin, polypeptide and combination thereof.In another exemplary embodiment, fiber comprises three kinds of different polymer or the subunit that is selected from following component: aliphatic polyester, polyalkylene oxide, polydimethylsiloxane, polyvinyl alcohol, polylysine, collagen, laminin, fibronectin, elastin laminin, alginate, fibrin, hyaluronic acid, mucin, polypeptide and combination thereof.In an exemplary embodiment, this aliphatic polyester is a straight chain or ramose.In another exemplary embodiment, this straight chain aliphatic polyester is selected from as follows: lactic acid (D-or L-), lactide, poly-(lactic acid), poly-(lactide), hydroxyacetic acid, poly-(hydroxyacetic acid), poly-(Acetic acid, hydroxy-, bimol. cyclic ester), Acetic acid, hydroxy-, bimol. cyclic ester, poly-(lactide-copolymerization-Acetic acid, hydroxy-, bimol. cyclic ester), poly-(lactic acid-copolymerization-hydroxyacetic acid), polycaprolactone and combination thereof.In another exemplary embodiment, this aliphatic polyester be ramose and comprise be selected from following at least a: be connected to the lactic acid (D-or L-) of junctional complex or biomolecule, lactide, poly-(lactic acid), poly-(lactide), hydroxyacetic acid, poly-(hydroxyacetic acid), poly-(Acetic acid, hydroxy-, bimol. cyclic ester), Acetic acid, hydroxy-, bimol. cyclic ester, poly-(lactide-copolymerization-Acetic acid, hydroxy-, bimol. cyclic ester), poly-(lactic acid-copolymerization-hydroxyacetic acid), polycaprolactone and combination thereof.In another exemplary embodiment, wherein said polyalkylene oxide is selected from as follows: poly(ethylene oxide), Polyethylene Glycol, poly(propylene oxide), polypropylene glycol and combination thereof.
[0142] in certain embodiments, this cellulosic polymers support is made up of single continuous fiber.In other embodiments, this cellulosic polymers support is by at least two, three, and four, or five kinds fibrous.In an exemplary, the quantity of the fiber in this cellulosic polymers support is selected from 2-100,000.In an exemplary, the quantity of the fiber in this cellulosic polymers support is selected from 2-50,000.In an exemplary, the quantity of the fiber in this cellulosic polymers support is selected from 50,000-100,000.In an exemplary, the quantity of the fiber in this cellulosic polymers support is selected from 10-20,000.In an exemplary, the quantity of the fiber in this cellulosic polymers support is selected from 15-1,000.
[0143] this cellulosic polymers support can comprise the fiber of at least a compositions.In an exemplary embodiment, this cellulosic polymers support comprises many dissimilar fibers, and this number is selected from 1,2, in 3,4,5,6,7,8,9 and 10 one.
[0144] in another exemplary embodiment, one or more fibers of cellulosic polymers support are biodegradable.In another exemplary embodiment, the fiber of this cellulosic polymers support comprises biodegradable polymer.In another exemplary, this biodegradable polymers comprises the monomer that is selected from lactic acid and hydroxyacetic acid.In another exemplary, this biodegradable polymers is poly-(lactic acid), poly-(hydroxyacetic acid) or its copolymer.Preferred biodegradable polymers is to be used for those materials of clinical use, for example poly-(lactic acid) and gather (hydroxyacetic acid) by FDA's approval.In another exemplary embodiment, biodegradable polymers support of the present invention can be used for guiding the form of engineering tissue to form and degraded gradually after this tissue set.The degradation rate of this polymer can be adjusted by those skilled in the art and be complementary with the production rate with this tissue.For example, if the polymer of fast degraded biologically is required, can select about 50:50
The combination of PLGA.Other modes that increase the polymer support biological degradability (for example comprise the more hydrophilic copolymer of selection, Polyethylene Glycol), reduce the molecular weight of polymer, because higher molecular weight means slower degradation rate usually, with change porosity or fibre density, because higher porosity and lower fibre density cause bigger water absorption rate and degraded faster usually.In another exemplary embodiment, this tissue is optional from as follows: muscular tissue, vascular tissue, nervous tissue, myeloid tissue and skin histology.In another exemplary embodiment, the Biodegradable fibers support can be used for guiding the form formation of through engineering approaches muscular tissue and sarcoplast, degrades gradually after myotube and skeletal muscle muscular tissue are assembled.
The manufacture method of cellulosic polymers support
[0145] polymer support of the present invention can be made in many ways.In an exemplary embodiment, this polymer support can be by the electrostatic spinning manufacturing.Electrostatic spinning is a kind of atomization process of conductor fluid, and it utilizes the interaction between electrostatic field and this conductor fluid.When the exterior static field puts on conductor fluid (for example, half dilute polymer solution or polymer melt), formed the conical microdroplet that suspends, thus the surface tension of this microdroplet and this electric field balance.Enough by force when overcoming the surface tension of this liquid, electrostatic atomization takes place at this electrostatic field.This drop becomes unstable and tiny jet and penetrates from the surface of this microdroplet then.When it arrives the ground connection target, this material can be collected as interconnected net, this net contains tiny relatively, i.e. small diameter fibers.The thin film (or film) that is obtained by these small diameter fibers has the ratio of very large surface area and volume and little aperture size.Electrostatic spinning equipment is described in detail in Zong, et al., Polymer, 43 (16): 4403-4412 (2002); Rosenet al., Ann Plast Surg., 25:375-87 (1990) Kim, K., Biomaterials2003,24, (27), 4977-85; Zong, X, Biomaterials 200,26, and (26) provide among the 5330-8.After electrostatic spinning, can utilize expressing technique and this polymer of the further molding of mold technology.For fibrous tissue being adjusted to aligned cellulosic polymers support, utilize patterned electrodes, curled hair tube colelctor electrode, or the post-treatment method for example uniaxial tension can be successful.Zong，X.，Biomaterials?2005，26，(26)，5330-8；Katta，P.，N?ano?Lett?2004，4，(11)，2215-2218；Li，D.，Nano?Lett?2005，5，(5)，913-6。
[0146] a kind of manufacturing polymer solution in can several modes.A kind of method comprises dissolves the polymer that is produced with monomer polymerization with in appropriate solvent.This process can realize in ejection assemblies or it can be loaded in the ejection assemblies subsequently.Another kind method comprise buy commercial polymer solution or commercial polymer and with their dissolving to generate polymer solution.For example, PLL can be from DuPont (Wilmington, DE), poly-(lactide-copolymerization-Acetic acid, hydroxy-, bimol. cyclic ester) can be from Ethicon (Somerville, NJ) and Birmingham Polymers (Birmingham AL) buys Sigma-Aldrich (St.Louis, MO) and Polysciences (Warrington PA) buys.Other manufacturers comprise LactelAbsorbable Polymers (Pelham, AL).Other polymer support components of the present invention, for example cell and biomolecule also are commercial, can available from supplier for example Invitrogen (SDiego, CA), Cambrex (Walkersville, MD), Sigma-Aldrich, Peprotech (Rocky Hill, NJ), R ﹠amp; D Systems (Minnea polis, MN), ATCC (Manassas, VA), Pierce Biotechnology (Rockford, IL).
[0147] polymer that is used to form polymer support at first is dissolved in solvent.This solvent can be any solvent, and this solvent can dissolve this polymer monomer and/or subunit and the polymer solution that can conduct electricity with electrostatic spinning is provided.Typical solvent comprises and is selected from following solvent: N, dinethylformamide (DMF), oxolane (THF), dichloromethane, dioxane, ethanol, hexafluoroisopropanol (HFIP), chloroform, water and combination thereof.
[0148] this polymer solution can be chosen wantonly to contain and produce the excessive charge work in order to promote the salt of this electrostatic spinning process.The example of the salt that is fit to comprises NaCl, KH
2PO
4, K
2HPO
4, KIO
3, KCl, MgSO
4, MgCl
2, NaHCO
3, CaCl
2Or the mixture of these salt.
[0149] polymer solution that forms this conductor fluid preferably has the polymer concentration of the about 80wt% of about 1wt%-, the more preferably from about about 60wt% of 8wt%-.Conductor fluid preferably has the viscosity of the about 2000mPXs of about 50mPXs-, more preferably from about the about 700mPXs of 200mPXs-.
[0150] electric field that produces in the electrostatic spinning process is preferably in the scope of the about 100kV of about 5kV-, more preferably from about the about 50kV of 10kV-.This conductor fluid is fed to the speed of spinning head (or electrode) preferably in the scope of about 0.1 mul/min-Yue 1000 mul/min, more preferably in about 1 mul/min-Yue 250 mul/min.
[0151] single or multiple spinning heads are positioned on the platform, and this platform is adjustable, change the distance between platform and the grounded collector base material.This distance can be any permission solvent in polymer and the distance of evaporation substantially fully before the grounded collector base material contacts.In an exemplary embodiment, this distance can change between the 25cm at 1cm.The distance that increases between grounded collector base material and this platform can be made thinner fiber usually.
[0152] under the situation of the electrostatic spinning of needs rotations mandrel, this mandrel and motor are usually by the drill chuck mechanical connection.In an exemplary, this motor is with the speed rotation mandrel of the about 500rpm of about 1rpm-.In another exemplary, this motor is with the speed rotation mandrel of the about 500rpm of about 200rpm-.In another exemplary, this motor is with the speed rotation mandrel of the about 100rpm of about 1rpm-.
[0153] this paper has described other embodiments or the modification that is used for electrostatic spinning process and equipment.
The variation of the electricity/mechanical performance of conductor fluid
[0154] character by the gained film of electrostatic spinning manufacturing is influenced by the electrical property of this conductor fluid and mechanical performance.The conductivity of this polymer solution can acutely change by/organic compound inorganic by the interpolation ion.The magnetohydrodynamics character of this polymer solution can be dependent on the combination of physical and mechanical property, (for example, the surface tension of liquid, viscosity and viscoelasticity) and electrical property (for example, the charge density of liquid and polarizability).For example, by adding surfactant in polymer solution, can reduce the tension force of flow surface, so that this electrostatic field can influence this jet shape and this jet in wideer condition and range.Can be by coupling connection with flow speed control under the constant pressure or the ejector pump under constant flow rate, the influence that can alleviate this conductor fluid viscosity.
Electrode design
[0155] is used for making the embodiment of film of the present invention at another, during electrostatic spinning, further optimizes the jet forming process and fiber size is better controlled to provide.Be not charged spinning head and the earth plate of only providing as discussed above, the battery lead plate that the spinning head of positively charged still is responsible for the formation of this polymer solution microdroplet and the center has a little outlet opening is responsible for this effusive formation.This outlet opening provides the instrument that makes jet pass this battery lead plate.Therefore, be positioned at place, can form rational electrostatic potential apart from the about 10mm of this spinning head if this polymer droplets on the spinning head of positively charged has typical sizes and this battery lead plate of 2-3mm.Short distance between two electrodes means that electrostatic potential can be quite low.But the electric field intensity of this gained can be enough strong for the electrostatic spinning process.By changing the electromotive force of spinning head, may command and the effusive formation of adjusting.Such electrode structure will significantly reduce the applying electrical potential that needs on the spinning head, typically, drop to about 1.5-2kV (with respect to the ground plate potential) from about 15kV.Form required definite spinning head electromotive force and depend on the fluidic electricity/mechanical performance of particular conductivity stablizing jet.
The control that jet quickens and carries
[0156] make in the polymer support embodiment preferred of the present invention at another, the jet range also is accurately to control.Jet by this battery lead plate outlet opening is a positively charged.Though this fluid has the tendency of self aligning when flight, under no external electrical field restriction, it is unstable that jet becomes on its path very soon.In other words, this charged fluid begins to scatter, and causes the microcosmic of convection cell and macroscopic properties out of hand.This unstability can by battery lead plate and a series of (etc.) use after the spaced electrode plate the careful probe electrode that is provided with to remove immediately.This electrode assemblie (or combination electrode), that is, probe electrode and battery lead plate can produce along the uniform distribution of (straight) flight path electrostatic potential.Place highlyer by approximately+20 to+30kV than target (ground potential) by the basic electromotive force with spinning head, the basic electromotive force that the electrostatic potential of regulating probe electrode simultaneously is lower than this battery lead plate forms accelerating potential.This combination electrode can be delivered to jet required target area.This combination electrode also will be used to operate this jet.By changing electrostatic potential, change effusive acceleration, cause the vary in diameter of the polymer fiber that forms.This electrostatic potential changes and has changed effusive stability, and therefore, the corresponding change of combination electrode can be used for stablizing new jet.These programs are used in the fibre diameter of finely tuning and changing during the electrostatic spinning process.
Spraying
[0157] in another embodiment, jet can by use be widely used in high-energy physics accelerator art " alternating gradient " (AG) technology focus on.Basic thought is to use two pairs of electrostatic quadrupole lenses.Second lens have the geometry arrangement identical with first lens and have (alternative) electrical gradient of counter-rotating.The jet of positively charged will be assembled, and for example, in the xz plane after first lens, reassembles in the yz plane after second lens then.The attention Z-direction is represented the direction of initial flight path.By additional triangular waveform signal being applied to a pair of electromotive force on quadrupole, this injection can sweep across the target area, and it is Guaranteed that effusive direction is in.In addition, because " sweeping across " electric potential signal waveform that changes can form required pattern on target.
Electrostatic spinning equipment with independent mandrel
[0158] the electrostatic spinning polymer fiber can be deposited on the base material of static or rotation.In the past, the stationary metal colelctor electrode is used for the random deposition of electrostatic spinning fiber.During electrostatic spinning, use the rotating metallic mandrel.The rotating metallic mandrel causes the random deposition of fiber on the mandrel surface, and it can make pipe when removing mandrel.Pipe with circumference fiber alignment alignment also can be by modifying this method and high speed (〉 100rpm) rotate mandrel and make.If the rotary drum of major diameter and/or length is as the colelctor electrode base material, non-aligned and aligned cellulosic polymers support thin slice can be by cutting and remove sedimentary cellulosic polymers support and make from this rotary drum.Also be presented at the inner air gap (hole) of making of stationary metal colelctor electrode before and brought out arrangement (Li, D., the Wang Y that passes the sedimentary electrostatic spinning fiber in gap.LL，Xia，Y。N., Electrospinning of Polymeric and Ceramic Nanofibers asUniaxially Aligned Arrays.Nano?Letters，2003。3(8)：p。1167-71)。But, at Li, or in any other document, all not to the manufacturing of aligned three-dimensional pipe or bar or the electrostatic spinning pipe that directly constitutes by vertical aligned fiber or the description of bar.
[0159] in yet another aspect, present invention resides on the rotation mandrel the method for aligned cellulosic polymers support electrostatic spinning.These cellulosic polymers supports can the required any orientations of user.In an exemplary, the vertical substantially or basic circumferential arrangement of this support.It also can be seamless that the cellulosic polymers support that produces by this method both can have seam.In an exemplary embodiment, the cellulosic polymers support is seamless.In another exemplary, the cellulosic polymers support is being seamless on the direction substantially parallel with this polymer support longitudinal axis.
[0160] in yet another aspect, the present invention includes the permission seamless pipe, the independent mandrel of seamless filled pipe and seamless bar electrostatic spinning.In another exemplary, this seamless pipe, seamless filled pipe and seamless bar have non-aligned fibre orientation.In another exemplary, this seamless pipe, seamless filled pipe, the fiber of seamless bar is aligned.In another exemplary, this seamless pipe, seamless filled pipe, seamless bar have basic vertically aligned fiber.
[0161] in yet another aspect, the present invention includes and allow integrally formed pipe, fill the independent mandrel of pipe and bar electrostatic spinning.In another exemplary, this integral body forms pipe, fills pipe and bar and has non-aligned fibre orientation.In another exemplary, this integral body forms pipe, and the fiber of filling pipe and bar is aligned.In another exemplary, this integral body forms pipe, fills pipe and bar and has basic vertically aligned fiber.
[0162] in an exemplary, mandrel with can be that the engine device of center rotation mandrel is connected with its longitudinal axis.In electrostatic spinning equipment, this rotation mandrel be ground connection and be positioned at spinning head below.Polymer solution is delivered to the most advanced and sophisticated of spinning head and makes charged by power supply.The electric field that produces between spinning head and mandrel brings out the vertical electropolymer solution of spinning head and forms injection.This injection is sprayed to the mandrel direction.One conduction region of this polymer contact mandrel contacts second conduction region of this mandrel then, passes this mandrel non-conductive area or air gap deposition.This causes having formed aligned fiber laydown on non-conductive area or air gap.By the rotation mandrel, obtain the even overlay of aligned fiber.The shape of the air gap between this deposit fiber layer and mandrel or the mandrel is consistent, is therefore forming thin slice in some cases, is bar under in some cases for pipe or the situation at other.Comprise thin slice, the fiber of pipe or bar can be arranged along the length direction of pipe or bar, has therefore formed the thin slice with vertical aligned fiber, pipe or bar.In an exemplary embodiment, this pipe or bar are seamless.In another exemplary, this pipe or bar are seamless along the direction substantially parallel with the major axis of this pipe or bar.
[0163] in one embodiment, the invention provides mandrel with at least two conduction regions and at least one non-conductive area.Such mandrel can design in many ways; The exemplary description of this mandrel is provided in Fig. 3 B and is described in Fig. 1 as the description of the mandrel of making a thin slice of the present invention and/or a tube apparatus part, and 2, among 2A and the 2B.In an exemplary embodiment, conductive material is a metal.In another exemplary, this metal is selected from steel and aluminum.Under a kind of situation, the zone of conduction mandrel can be covered by electrically non-conductive material.The exemplary cross section of this mandrel is provided in Fig. 3 D.In an exemplary, this electrically non-conductive material is selected from as follows: adhesive tape, insulating tape, politef, and plastics.Under the another kind of situation, can make and have at least three parts, the non-conductive area in two conductions of interconnection mandrel districts.Under another situation, the discontinuous part of non-conductive area between two conduction mandrel districts, extending.The exemplary cross section of this mandrel is provided in Fig. 3 C.Additional non-conductive area can be by placing the non-conductive area on the conduction region of this mandrel, or add on this mandrel by the non-conductive area that is interconnected between two conduction regions of mandrel.These additional non-conductive areas if be used in combination with additional spinning head, can promote the production of a more than pipe on identical mandrel simultaneously.In one embodiment, the invention provides mandrel with at least three conduction regions and at least two non-conductive areas.In one embodiment, the invention provides mandrel with at least four conduction regions and at least three non-conductive areas.In one embodiment, the invention provides mandrel with at least five conduction regions and at least four non-conductive areas.
[0164] in one embodiment, the invention provides and have first conduction region, second conduction region and the mandrel of air gap between first conduction region and second conduction region.Such mandrel can design in many ways; The exemplary description of this mandrel is provided in Fig. 3 E.This mandrel is described in Fig. 6 as the exemplary description of the part of production bar apparatus of the present invention, and 7, and 7A.The embodiment with bull spinning head that provides is described among Figure 11.In an exemplary embodiment, conductive material is a metal.In another exemplary, this metal is selected from steel and aluminum.In an exemplary, each conduction region of mandrel and another are arranged in rows.In an exemplary, each conduction region of mandrel with can connect with the assembly of identical speed rotation.This can turn round under identical speed with each motor of assurance by each conduction region that electric motor assembly is connected to mandrel and realize.This also can be by guaranteeing mandrel each conduction region be communicated with identical motor and realize.
[0165] after electrostatic spinning is finished, polymer support of the present invention is removed from mandrel.For thin slice support polymer, this thin slice can be peeled off from mandrel.For the pipe polymer support, this mandrel can take out in electric motor assembly, shifts out pipe then.In some embodiments, shift out also can be by taking apart or yet can realize by the cutting pipe at intermediary mandrel.For the bar polymer support, this bar can be peeled off from the metal end of conduction region.Under some situation, the length of this deposit fiber is not equal to, and causing in the one or both ends of polymer support is jagged edge.Randomly, in order to produce polymer support, can cut an end of this polymer support with basic identical dimensions length.When polymer support is on mandrel, or at it after mandrel is removed, can carry out such cutting.
[0166] feature of this polymer support described herein can change by changing a plurality of parameters.For example.SOME METHODS is arranged, and it both can use separately also and can be used in combination, and can reduce the average diameter of fiber in the cellulosic polymers support.A kind of method is to add more salt in this polymer solution.In polymer solution, use the stronger solvent of polarity also to be easy to reduce fiber diameter, between spinning head and mandrel, increase distance and also play same effect.Reduce the concentration that the stent diameter additive method comprises the voltage of increase equipment and increases polymer.
[0167] the multiple layer polymer support can be formed by the method for finishing several mandrel rotations by as herein described.For example, multilayer pipe can be formed by the rotation of finishing several mandrels.Also can make other have more than one type the layer polymer support.In an exemplary, the circumferential arrangement of this fiber also can be regulated or changes by the speed that changes the mandrel rotation.Therefore can make polymer support with inner vertically alignment layer and exterior periphery alignment layer.In order to make multilamellar hollow pipe support, can use various mandrels and rotating speed with each layer of particular arrangement.In an exemplary, hollow tubular fibroid support is fabricated to fibrous outer with vertically aligned chamber layer and circumferential arrangement alignment.A kind of method of making such support comprises uses foregoing mandrel with non-conductive area.This mandrel low speed rotation is to allow forming by vertical aligned fibrous tube fiber support.Increase the rotating speed of mandrel then, it causes that the electrostatic spinning fiber centers on the circumferential arrangement of vertically aligned fibroid pipe on the edge.In another exemplary, internal layer vertically arrange and should skin fibrous by the random alignment alignment.This can realize by same apparatus of describing before using, except when form when outer, this mandrel with prevent fiber vertically and the middling speed of circumferential arrangement alignment rotate.
II.b) polymer support of micro-patterning
[0168], the invention provides the compositions of the polymer support that comprises micro-patterning in second aspect.Because micro-patterning, soft etching technique are used for the space of shape or this polymer of chemical modification and organize for how much to generate the micro-meter scale feature at substrate surface.Taylor，A。M.，NatMethods?2005，2，(8)，599-605；Dow，J。A.，J?Cell?Sci?Suppl1987，8，55-79；Kane，R。S.，Biomaterials?1999，20，(23-24)，2363-76。The polymer support that is generated by these technology can be used for controlling many aspects of cell behavior, comprises cell size, shape, spatial organization, propagation and existence.Chen，C.S.，Science?1997，276，(5317)，1428-8；Bhatia，S.N.，Faseb?J1999，13，(14)，1883-900；Deutsch，J.，J?Biomed?Mater?Res2000，53，(3)，267-76；Folch，A.，nnu?Rev?Biomed?Eng?2000，2，227-56；Whitesides，G.M.，Annu?Rev?Biomed?Eng?2001，3，335-73。Poly-(dimethyl siloxane) is a kind ofly can have high reproducibility by micro-patterning (PDMS), and can be the elastomer that cell connects provides flexible substrate.Wang，N.，CellMotil?Cytoskeleton?2002，52，(2)，97-106。
II.c) arrangement of polymer support
[0169] polymer support of the present invention can have oriented or random orientation.In oriented, at least 50% fiber that comprises polymer support is along arranging all axle orientations.
[0170] in an exemplary, said composition has and is selected from following arrangement: substantially longitudinally, basic circumference with decussation.When fiber along pipe, fill pipe or shaft-like polymer support long axis direction when arranging, exist vertically and arrange.When fiber is when the polymer support short-axis direction is arranged, there is circumferential arrangement.When a kind of polymer support fiber in the compositions is arranged with respect to equal angled mode of second polymer support (itself and first polymer support adjacency) arrangement with the equal axle of first polymer support arrangement, have the arrangement of decussation, vertically the polymer support of arrangement or circumferential arrangement alignment can have the fiber of more than one deck.Decussation arranged polymeric support needs the fiber of more than one deck.
[0171] in another exemplary, polymer fiber has the standard deviation with the fibre bundle central shaft.In an exemplary, the standard deviation of described fiber is selected from as follows: about 0 °-Yue 1 °, and about 0 °-Yue 3 °, about 0 °-Yue 5 °, about 0 °-Yue 10 °, about 0 °-Yue 15 °, about 0 °-Yue 20 ° and about 0 °-Yue 30 °.
[0172] arrangement of the polymer support pair cell skeleton of Pai Lieing, cell migration and cell function have deep effect.The polymer support of arranging can be induced the regeneration that therefore improves tissue with the cell guiding migration.Such support is to solve the multiple for example muscle of organizing, vascular tissue, the neural and the most promising method of spinal cord regeneration.For example, vertically aligned cellulosic polymers support can improve and the particular pilot nerve, skin, and the damage interstital growth passes in muscle and/or vascular tissue.
[0173] direction that is positioned at of the polymer support of wherein arranging can influence the biological function of replacing or improving the polymer support of arranging.For example, when the arranged polymeric support is positioned at wound, wound healing is faster, this moment aligned polymer support perpendicular to rather than be parallel to the major axis of wound.In an exemplary, the central longitudinal axis of the polymer support that this bundle is aligned is positioned at perpendicular to the direction of improving or replacing the material of the polymer support of arranging.In another exemplary, the central longitudinal axis of the polymer support that this bundle is aligned is positioned at and the parallel direction of material direction of improving or replacing the arranged polymeric support.
[0174] in another exemplary, the compositions of arrangement of the present invention (for example polymer support) can comprise biodegradable polymers.The form that these compositionss can be used for guiding other types to have the anisotropic structure tissue forms, for example, and nerve, skin, blood vessel, skeletal muscle, cardiac muscle, tendon and ligament.These arrange the present invention, and biodegradable compositions also can be used for the formation of three-dimensional tissue.Use the biodegradable cellulosic polymers support of electrostatic spinning, can generate nervous tissue, myeloid tissue, skin tissue, the three-dimensional structure of vascular tissue and muscular tissue.
[0175] in an exemplary, compositions described herein can comprise a more than polymer support.Each of those polymer supports can have with compositions in the identical or different arrangement of other one or more polymer supports.
[0176] in an exemplary, said composition comprises two polymer supports.First polymer support has tube shape and is vertically aligned.Second polymer support centers on the outside of first polymer support and has the orientation that is selected from following orientation: at random, and circumference, decussation and vertical.In an exemplary, the orientation of second polymer support is selected from random and circumference.
II.D) method of the shape of polymer support/manufacturing polymer support
[0177] polymer support of the present invention can form multiple shape, depends on the character of dealing with problems.
[0178] compositions of the present invention and/or polymer support have various sizes.In an exemplary, polymer support is that 0.1mm-50cm is long.In each and every one exemplary, this polymer support is that 0.1mm-1mm is long at another.In another exemplary, this polymer support is that 1mm-1cm is long.In another exemplary, this polymer support is that 1cm-10cm is long.In another exemplary, this polymer support is that 10cm-50cm is long.In another exemplary, this polymer support is that 1cm-5cm is long.In another exemplary, this polymer support is that 2.5cm-15cm is long.In another exemplary, this polymer support is that 5mm-6cm is long.In another exemplary, this polymer support is that 8mm-3cm is long.In another exemplary, this polymer support is that 10cm-25cm is long.In another exemplary, this polymer support is that 0.5cm-2cm is long.In another exemplary, this polymer support is that 0.1cm-2cm is long.
[0179] present composition and/or polymer support can be made up of various fibrous layers.In an exemplary, said composition has about 2,000 fibrous layers of about 1-.In an exemplary, said composition has about 1,000 fibrous layer of about 1-.In an exemplary, said composition has about 500 fibrous layers of about 1-.In an exemplary, said composition has about 20 fibrous layers of about 1-.In an exemplary, said composition has about 10 fibrous layers of about 1-.In an exemplary, said composition has about 25 fibrous layers of about 5-.In an exemplary, said composition has about 1,500 fibrous layer of about 500-.In an exemplary, said composition has about 20 fibrous layers of about 10-.In an exemplary, said composition has about 80 fibrous layers of about 35-.In an exemplary, said composition has about 100 fibrous layers of about 10-.In an exemplary, said composition has about 600 fibrous layers of about 5-.In an exemplary, said composition has about 80 fibrous layers of about 10-.In an exemplary, said composition has about 12 fibrous layers of about 2-.In an exemplary, said composition has about 400 fibrous layers of about 60-.In an exemplary, it is about 1 that said composition has, about 1,750 fibrous layer of 200-.
[0180] in an exemplary, polymer support has the shape of thin slice or film.The polymer support film can be made by electrostatic spinning.Each fiber in film inside can be arranged during using rotary drum as the electrostatic spinning of colelctor electrode or after mechanical uniaxial tension.
[0181] in another exemplary, this polymer support has cross-shaped.In order to form the decussation thin slice, but the layer of arranged polymeric thin slice or film can be arranged to be selected from as the angle of lower angle each other: greater than 20 degree less than 160 degree, greater than 30 degree but less than 150 degree, greater than 40 degree but less than 140 degree, greater than 50 degree but less than 130 degree, greater than 60 degree but less than 120 degree, greater than 70 degree but less than 110 degree with greater than 80 degree but less than 100 degree.
[0182] variety of way of making the decussation thin slice is arranged.In an exemplary, use the rotating metallic rotary drum colelctor electrode that does not contain the non-conductive area.The fiber alignment layer generates on drum, then it is peeled off from drum.This alignment layer is revolved to turn 90 degrees and is put back to then on the drum.In this drum high speed rotating, add the next extra play of electrostatic fibre.Other decussation layers can add by repeating these steps.In another exemplary, use the rotary drum that the non-conductive area is arranged.Herein, this rotary drum slowly rotates the phase I and makes this fiber laydown and vertically be arranged in non-conducting portion.Thereby this this fiber of rotary drum fast rotational is forced to circumferential arrangement then.Other decussation layer can add by repeating these steps.
Pipe
[0183] in another exemplary, polymer support has tubular.The exemplary description that the exemplary description of pipe is provided in the cross-sectional view strength of Fig. 4 A and pipe is provided in Fig. 5 A.Pipe can have various sizes, depends on its length, with and internal diameter and external diameter.In an exemplary, the essentially no cellulosic polymers support in the inner space of this pipe.Can change these parameters to adapt to, for example, the size of multiple tissue and application.In an exemplary embodiment, this tube wall is by aligned fibrous.In another exemplary, this fiber is vertically arranged or circumferential arrangement.In another exemplary embodiment, this pipe has seam.In another exemplary, the seam of this pipe is basic parallel with the longitudinal axis of pipe.In another exemplary embodiment, this pipe is seamless.In another exemplary, seamless substantially on the longitudinal axis parallel direction of this Guan Zaiguan.In another exemplary, the inwall of this pipe form by vertical aligned fibrous layer and the outer wall of pipe by non-aligned fibrous.In another exemplary, the pipe parallel with the longitudinal axis of this pipe be the inwall of seamless and this pipe form by vertical aligned fibrous layer and the outer wall of this pipe by non-aligned fibrous.The pipe of Xian Dinging is because existence demonstrates bigger structural intergrity as outer sheath shell randomly oriented fiber in this case.In another exemplary, the outer wall of the inwall of pipe pipe by the fibrous of non-aligned random orientation is made up of vertical aligned fibrous layer.In another exemplary, this pipe parallel with the pipe longitudinal axis is seamless.In another exemplary, the inwall of this pipe is by fibrous by the circumferential arrangement alignment of the outer wall of vertical aligned fibrous and pipe.In another exemplary, the pipe of the direction that this is substantially parallel with the pipe longitudinal axis is seamless.
[0184] in an exemplary, the distance between the inner and outer wall of this pipe is about 1nm to 50,000nm.In another exemplary, the distance between the inner and outer wall of this pipe is about 1nm to 10,000nm.In another exemplary, the distance between the inner and outer wall of this pipe is about 1nm to 5,000nm.In another exemplary, the distance between the inner and outer wall of this pipe is that about 1nm is to 500nm.In another exemplary, the distance between the inner and outer wall of this pipe is that about 1nm is to 50nm.In another exemplary, the distance between the inner and outer wall of this pipe is that about 1nm is to 5nm.In another exemplary, the distance between the inner and outer wall of this pipe is that about 10nm is to 500nm.In another exemplary, the distance between the inner and outer wall of this pipe is about 100nm to 1,000nm.In another exemplary, the distance between the inner and outer wall of this pipe is about 5,000nm to 15,000nm.In another exemplary, the distance between the inner and outer wall of this pipe is about 20,000nm to 50,000nm.In another exemplary, the distance between the inner and outer wall of this pipe is that about 75nm is to 600nm.In another exemplary, the distance between the inner and outer wall of this pipe is about 2,000nm to 7,000nm.
[0185] in an exemplary, the internal diameter of this pipe is about 1nm to 50,000nm.In another exemplary, be about 1nm to 10 at the internal diameter of this pipe, 000nm.In another exemplary, be about 1nm to 5 at the internal diameter of this pipe, 000nm.In another exemplary, the internal diameter of this pipe for about 1nm to 500nm.In another exemplary, the internal diameter of this pipe for about 1nm to 50nm.In another exemplary, the internal diameter of this pipe for about 1nm to 5nm.In another exemplary, the internal diameter of this pipe for about 10nm to 500nm.In another exemplary, be about 100nm to 1 at the internal diameter of this pipe, 000nm.In another exemplary, be about 5 at the internal diameter of this pipe, 000nm to 15,000nm.In another exemplary, be about 20 at the internal diameter of this pipe, 000nm to 50,000nm.In another exemplary, the internal diameter of this pipe for about 75nm to 600nm.In another exemplary, be about 2 at the internal diameter of this pipe, 000nm to 7,000nm.
[0186] pipe as herein described can be made in many ways.In an exemplary embodiment, this pipe is not made by electrostatic spinning.In another exemplary, this pipe is by unoriented fiber or unoriented at random polymer support are formed at random.
[0187] in an exemplary, the pipe that cellulosic polymers support thin slice roll compacting manufacturing is had seam.At first, cellulosic polymers support thin slice is made by electrostatic spinning.The fiber that comprises thin slice can be arranged during electrostatic spinning.Make some method of arranging the electrostatic spinning fiber and comprise that the use rotary drum is as the method for grounded collector base material or by using the method for mandrel described herein.In an exemplary embodiment, this mandrel is mandrel 56A.The fiber that comprises thin slice also can be arranged by mechanical uniaxial tension after electrostatic spinning.The roll compacting around mandrel of aligned then cellulosic polymers support thin slice is managed to form.This mandrel both can be before having fixed this pipe also can after remove.In an exemplary, two ends of thin slice that will be parallel with the polymer support longitudinal axis are fixed together to make vertically aligned seamed pipe then.In another exemplary, the roll compacting around this mandrel of this thin slice is fixed on the part of pipe to generate vertically aligned seamed pipe with an end of this thin slice more than once.By annealing (heat), bonding or sew up and to realize that this fixes.The example of binding agent comprises solvent or for example fibrin sealant and the collagen gel agent of binding agent biology.
[0188] now will be in detail with reference to several embodiments of the present invention, its example is example in the accompanying drawings.Although the present invention will describe in detail in conjunction with embodiment subsequently, should be appreciated that they are not intended to limit the invention to these embodiments.On the contrary, the present invention is intended to cover the alternative that can be included in the spirit and scope of the present invention that limited by appended claim, modification and equivalence.
[0189] in another exemplary, the invention provides seamless pipe.Fig. 1 is for making the electrostatic spinning equipment 30 of this spline structure.Polymer solution 38, it comprises the polymer that is dissolved in solvent, is included within the ejection assemblies 36.This ejection assemblies 36 is the part of jet pump assembly 32, and wherein computer 34 comes controlling polymers solution to discharge the speed of this ejector by controlled pressure or flow velocity.Randomly, can provide and control different flow velocitys selected spinning head.Depend on the required physical property of this polymer support and change flow velocity, that is, and the thickness of film, fibre diameter, aperture size, the density of film etc.
[0190] to spinning head 42, this spinning head 42 is positioned at platform 44 to this eductor pump assembly 32 with feed of polymer solution.This spinning head is pointed, and it allow to spray forms and send and noiseless.Utilize high voltage power supply 48 by electric wire 41A will the about 30kV of about 10kV-voltage be applied on this spinning head.
[0191] mandrel 56A (it is comprised 55 as what Fig. 3 B mentioned, 57A and 57B) thus below spinning head 42, between charged spinning head and mandrel 56A, form electric field.This electric field makes a branch of polymer solution from the ejection of this spinning head and be ejected into mandrel 56A, forms the filament or the fiber 46 of micron or nanometer diameter.This drill chuck uses earth lead 41B and 41C ground connection.
[0192] this mandrel 56A connects first drill chuck 54 (being connected to non-conductive bearing 60) and the second drill chuck 54A (being connected to non-conductive bearing 60A) that is connected with motor 52.This motor 52 is connected with speed control 50, the speed of this motor rotation mandrel of these speed control 50 controls 56A.Randomly, can provide different rotary speeies.Depend on the required physical property of this polymer support and change rotary speed, that is, and the thickness of film, fibre diameter, aperture size, the density of film etc.
[0193] in another exemplary embodiment, the invention provides seamless pipe by the device fabrication of Fig. 2 electrostatic spinning.This equipment class is similar to the equipment of Fig. 1, but also comprises the tower 40 that holds platform 44.
[0194] pipe with multiple layer polymer support can be made in many ways.In an exemplary, additional polymer support thin slice can be wrapped in the outer or pipe as herein described of pipe as herein described.In an exemplary, generate vertically aligned cellulosic polymers support, it is seamless or has seam.Non-then aligned micrometer/nanometer fiberoptic fiber polymer support thin slice be positioned at vertical arrangement pipe around have inner vertically aligned fibrous layer and outside non-aligned fibrolaminar bimetallic tube with generation.Pipe (3,4,5,6 etc.) with other numbers of plies is that these methods may be extended.Stitching or binding agent can randomly add this polymer to keep this structure.
[0195] in another exemplary, produce vertically aligned cellulosic polymers support, it is seamless or has seam.What then circumferential arrangement aligned fibers polymer support thin slice is placed vertical arrangement pipe has inner vertical aligned fibrous layer and outside non-aligned fibrolaminar bimetallic tube with formation on every side.Stitching or binding agent can at random add this polymer to keep this structure.
[0196] in another exemplary, generate seamless cellulosic polymers support tube with inner and outer wall, wherein inwall by vertically aligned fibrous and outer wall by circumferential arrangement align fibrous.During electrostatic spinning, use the mandrel that has two conduction regions in the both sides, non-conductive area described herein.This mandrel with low speed rotation to form the uniform deposition of vertical aligned fiber.This mandrel is with the uniform deposition of high speed rotating with formation circumferential arrangement aligned fibers then.Obtain seamless cellulosic polymers support tube, it has inner vertically aligned fibrous layer and the aligned fibrous layer of exterior periphery.
[0197] in another exemplary, generate seamless cellulosic polymers support tube with inner and outer wall, wherein inwall is non-aligned fibrous by circumference by vertically aligned fibrous and outer wall.The above-mentioned specific mandrel that has two conduction regions in the both sides, non-conductive area uses during electrostatic spinning.This mandrel with low speed rotation to form the uniform deposition of vertical aligned fiber.This mandrel rotates with middling speed then, and this speed prevents the arrangement of the vertical and circumference of deposit fiber.Obtain seamless cellulosic polymers support tube, it has inner vertically aligned fibrous layer and the aligned fibrous layer of random external.
Bar
[0198] in another exemplary, polymer support has the shape of bar.The exemplary description that the exemplary description of bar is provided in the cross-sectional view strength of Fig. 4 B and bar is provided in Fig. 5 B.Bar can have various sizes, depends on its length, with and diameter.Also can change the quantity of fiber in the bar, this will influence the density of this bar.Can change these parameters to adapt to, for example, the size of various tissues and application.In an exemplary embodiment, this bar is by aligned fibrous.In another exemplary, this fiber is vertically arranged or circumferential arrangement.In another exemplary embodiment, this bar has seam.In another exemplary, the seam of bar is basic parallel with the longitudinal axis of bar.In another exemplary embodiment, this bar is seamless.In another exemplary, this bar is seamless in the direction parallel with the bar longitudinal axis basically.In another exemplary, this bar comprises vertically aligned fibrous layer, and it is covered by non-aligned fibrous pipe.In another exemplary, this bar is seamless in the direction parallel with the bar longitudinal axis basically, and this bar comprises vertical aligned fibrous layer, and it is covered by non-aligned fibrous pipe.Owing to, the material that limits in this case is designed to demonstrate bigger structural intergrity as the existence of outer sheath shell randomly oriented fiber.In another exemplary, bar by non-aligned random orientation fibrous and this pipe by vertically aligned fibrous.In another exemplary embodiment, this bar is seamless in the direction parallel with the bar longitudinal axis basically.In another exemplary, this bar is by vertically aligned fibrous, and it is covered by the fibrous pipe by the circumferential arrangement alignment.In another exemplary embodiment, this bar is seamless in the direction parallel with the bar longitudinal axis basically.
[0199] in an exemplary, the about 1nm to 50 of the diameter of this bar, 000nm.In another exemplary, the diameter of this bar is about 1nm to 10,000nm.In another exemplary, the diameter of this bar is about 1nm to 5,000nm.In another exemplary, the diameter of this bar is that about 1nm is to 500nm.In another exemplary, the diameter of this bar is that about 1nm is to 50nm.In another exemplary, the diameter of this bar is that about 1nm is to 5nm.In another exemplary, the diameter of this bar is that about 10nm is to 500nm.In another exemplary, the diameter of this bar is about 100nm to 1,000nm.In another exemplary, the diameter of this bar is about 5,000nm to 15,000nm.In another exemplary, the diameter of this bar is about 20,000nm to 50,000nm.In another exemplary, the diameter of this bar is that about 75nm is to 600nm.In another exemplary, the diameter of this bar is about 2,000nm to 7,000nm.
[0200] bar as herein described can be made in many ways.In another exemplary embodiment, this bar is not made by electrostatic spinning.In another exemplary, this bar is by unoriented fiber or unoriented at random polymer support are formed at random.
[0201] in an exemplary, the bar that cellulosic polymers support thin slice roll compacting manufacturing is had seam.At first, cellulosic polymers support thin slice is made by electrostatic spinning.The fiber that comprises thin slice can be arranged during electrostatic spinning.Some method of making aligned electrostatic spinning fiber comprises the method for rotary drum as grounded collector base material or mandrel described herein of using.In an exemplary embodiment, this mandrel is mandrel 56B.The fiber that comprises thin slice also can be arranged by mechanical uniaxial tension after electrostatic spinning.This aligned cellulosic polymers support thin slice self roll compacting forms bar then.An end of this polymer support thin slice is fixed on the part of this bar to generate vertically aligned seam bar then.By annealing (heat), bonding or this thin slice is fixed together by suture.The example of binding agent comprises solvent or binding agent biology for example adhesive fibrin and collagen gel.
[0202] now will be in detail with reference to several embodiments of the present invention, its example is example in the accompanying drawings.Although the present invention will describe in detail in conjunction with embodiment subsequently, should be appreciated that they are not intended to limit the invention to these embodiments.On the contrary, the present invention is intended to cover the alternative that can be included in the spirit and scope of the present invention that limited by appended claim, modification and equivalence.
[0203] in another exemplary, the invention provides seamless bar.Fig. 6 is for making the electrostatic spinning equipment 80 of this spline structure.Polymer solution 38, it comprises the polymer that is dissolved in solvent, is included within the ejection assemblies 36.This ejection assemblies 36 is the part of jet pump assembly 32, and wherein computer 34 comes controlling polymers solution to discharge the speed of this ejector by controlled pressure or flow velocity.Randomly, can provide and control different flow velocitys selected spinning head.Depend on the required physical property of this polymer support and change flow velocity, that is, and the thickness of film, fibre diameter, aperture size, the density of film etc.
[0204] to spinning head 42, this spinning head 42 is positioned on the platform 44 this eductor pump assembly 32 with feed of polymer solution.This spinning head is pointed, and it allow to spray forms and send and noiseless.Utilize high voltage power supply 48 will about 10kV to be applied on this spinning head to the voltage of about 30kV by electric wire 41A.
[0205] mandrel 56B (it is mentioned as Fig. 3 C, comprises 57A, 57B and 58) is positioned at below the spinning head 42.This mandrel 56B has first conduction region 57 and the first conducting surface 57C, the second conduction region 57B and the second conducting surface 57D, thus between charged spinning head and this mandrel 56B, generate electric field.This electric field makes a branch of polymer solution from the ejection of this spinning head and be ejected into mandrel 56B, is formed on the micron in 58 or the filament or the fiber of nanometer diameter.Drill chuck uses earth lead 41B and 41C ground connection.
[0206] first conduction region 57A is connected to first drill chuck 54 (being connected to non-conductive bearing 60) and the second conduction region 57B is connected (being connected to non-conductive bearing 60A) with the second drill chuck 54A, and this second drill chuck 54A is connected with motor 52A.Motor 52 is connected with speed control 50A with 52A, the speed of this speed control 50A control motor rotation mandrel 56B.Randomly, can provide different rotary speeies.Depend on the required physical property of this polymer support and change rotary speed, that is, and the thickness of film, fibre diameter, aperture size, the density of film etc.
[0207] in another exemplary embodiment, the invention provides seamless bar by the device fabrication of Fig. 7 electrostatic spinning.This equipment class is similar to the equipment of Fig. 6, but also comprises the tower 40 that holds platform 44.
[0208] bar with multiple layer polymer support can be made in many ways.In an exemplary, additional polymer support thin slice can be wrapped in outside the bar as herein described.In an exemplary, generate vertically aligned cellulosic polymers support, it is seamless or has seam.The cellulosic polymers support thin slice of non-then aligned micrometer/nanometer fiber be positioned at vertical arrangement bar around have inner vertical aligned fibrous layer and outside non-aligned fibrolaminar ELECTRODE WITH BILAYER POLYMERIC thing support with generation.Pipe (3,4,5,6 etc.) with other numbers of plies is may extending of these methods.Sew up agent or binding agent and can randomly add this polymer to keep this structure.Some of these multilamellar bar embodiments also can be described as " filling pipe ".
[0209] in another exemplary, generate vertically aligned cellulosic polymers support, it is seamless or has seam.The cellulosic polymers support thin slice of circumferential arrangement alignment has inner vertical aligned fibrous layer and the aligned fibrolaminar double-deck bar of exterior periphery with formation around placing vertically aligned bar then.Stitching or binding agent can at random add this polymer to keep this structure.
[0210] in another exemplary, seamless cellulosic polymers support has inner shaft and exterior tube or sleeve pipe, wherein this inner shaft by vertically aligned fibrous and exterior tube or sleeve pipe by circumferential arrangement or random alignment align fibrous.The manufacturing as described herein of seamless vertical aligned cellulosic polymers shaft bar.For exterior tube or the sleeve pipe that forms the circumferential arrangement aligned fibers, the rotation of mandrel is increased at a high speed to allow to be centered around the uniform deposition of vertically aligned cellulosic polymers bar circumferential arrangement aligned fibers on every side.Perhaps, for the exterior tube or the sleeve pipe that form the random alignment aligned fibers, the rotation of mandrel is increased to middling speed, and this middling speed prevents the vertical and circumferential arrangement of sedimentary fiber on vertically aligned cellulosic polymers bar.When from mandrel, removing this cradling piece, obtain having inner shaft and exterior tube or telescopic seamless cellulosic polymers support, wherein this inner shaft by vertically aligned fibrous and exterior tube or sleeve pipe by circumferential arrangement and non-aligned fibrous.
Fill pipe
[0211] in an exemplary, this polymer support has the shape of filling pipe.This filling pipe can followingly be made: (1) formation pipe as described herein; (2) be used to fill the packing material of pipe by vertically aligned fibrous.This packing material can be loose, highly porous material.In an exemplary embodiment, this packing material is made the thin film of aligned fiber by electrostatic spinning.This material directly is inserted into pipe as herein described inside then, and it is with the direction orientation parallel with the major axis of this pipe.Under another situation, the manufacturing as described herein of vertical aligned fiber rod.Then this bar or: (1) directly be inserted into the pipe of abundant formation inner or (2) fill pipe with sewing up agent or adhesive seal to form then as the mandrel that centers on around sheets of fibres roll compacting.
II.f) additive composition or polymer support component
II.f1) cell
[0212] in an exemplary, compositions as herein described and/or polymer support further comprise cell.This cell can be on the surface of compositions and/or polymer support or embedding wherein or embed wherein.In an exemplary, this cell is covalently bound gets in touch to compositions of the present invention and/or polymer support or compositions non-covalent and of the present invention and/or polymer support.In certain embodiments, this cell is used to promote neoblastic growth.In an exemplary, this cell is selected from as follows: from (donor and receptor are individuals with same) of body, allochthonous (donor and receptor are not individuals with same, but come from identical species) and allogenic (donor comes from different species with receptor).In an exemplary embodiment, this cell is not a stem cell.In an exemplary embodiment, this cell is a stem cell.In an exemplary, this cell is selected from as follows: adult stem cell and embryonic stem cell.In another exemplary, adult stem cell can be a mescenchymal stem cell (MSC) (deriving from bone marrow) or derived from the adult stem cell of fatty tissue (ADAS).In another exemplary, this stem cell is selected from as follows: unipotency stem cell, versatility (multipotent) stem cell, versatility (pluripotent) stem cell and totipotency stem cell.In an exemplary embodiment, this cell is a CFU-GM.In another exemplary, CFU-GM can be a fibroblast, sarcoplast, neural progenitor cell, hemopoietic CFU-GM, and endothelial progenitor cells.In another exemplary, this cell is selected from sarcoplast and muscle CFU-GM.In another exemplary, this cell is selected from as follows: the myocyte that grows up, muscle CFU-GM, muscle stem cell or its combination.In another exemplary, this cell is selected from as follows: grow up vascular cell, blood vessel CFU-GM, blood vessel stem cell or its combination.In another exemplary, this cell is selected from as follows: grow up neurocyte, neurogliocyte, neural progenitor cell, neuroglia CFU-GM, neural stem cell, neuroepithelial cell or its combination.In another exemplary, this cell is selected from Schwann cell, fibroblast and vascular cell.In another exemplary, this cell is selected from as follows: the Skin Cell of growing up, skin CFU-GM, skin progenitor cell.Recently, be used in reference to the guided cell growth, the feasibility of the nano fiber basis material of function and tissue obtains proof fibroblast on vascular cell and the mescenchymal stem cell.Zong，X.，Biomacromoleucles?2003，4，(2)，416-23；Li?D.，AdvMater?2004，16，(4)，1151-1170；Boland，E。D.，Front?Biosci2004，9，1422-32；Bhattarai，S。R。Biomaterials?2004，25，(13)，2592-602；Yoshimoto，H.，B，iomaterials?2003，24，(12)207782。
[0213] in another embodiment, as herein described have the compositions of cell embedding and/or a development that polymer support can be used to three-dimensional tissue.In another exemplary, there is the polymer support of sarcoplast embedding to can be used for developing muscular tissue, the polymer support of neurocyte-embedding can be used for developing nervous tissue, the polymer support of vascular cell embedding can be used for developing vascular tissue, and the polymer support that the polymer support of cord cell embedding can be used for developing myeloid tissue and Skin Cell embedding can be used for developing skin histology.
[0214] cell can mix said composition and/or polymer support inside after electrostatic spinning or back-manufacturing.
II.f2) biomolecule
[0215] biomolecule (for example nucleic acid, aminoacid, sugar or lipid) can be covalently bound to compositions as herein described and/or polymer support or non-covalent compositions as herein described and/or the polymer support of being connected to.In an exemplary, described biomolecule is selected from as follows: acceptor molecule, extracellular matrix component or the biochemistry factor.In another exemplary, the biochemistry factor is selected from somatomedin and differentiation factor.In an exemplary, described biomolecule is selected from glycosaminoglycans and Dan Baijutang.In an exemplary embodiment, this biomolecule is selected from as follows: heparin, Heparan sulfate, heparan sulfate proteoglycan and combination thereof.
[0216] in another exemplary, first molecule (it may or may not be a biomolecule) is covalently bound to compositions of the present invention and/or polymer support.This first molecule can be used for and second bio-molecular interaction.In an exemplary, first molecule is that the junctional complex and second biomolecule are selected from as follows: acceptor molecule, the biochemistry factor, somatomedin and differentiation factor.In an exemplary embodiment, first molecule is selected from as follows: heparin, Heparan sulfate, heparan sulfate proteoglycan and combination thereof.In an exemplary, second biomolecule is selected from as follows: acceptor molecule, the biochemistry factor, somatomedin and differentiation factor.In another exemplary embodiment, first molecule is covalently bound by junctional complex, and in another exemplary embodiment, junctional complex comprises and is selected from following member: water-soluble polymer and non-soluble polymer.In another embodiment, junctional complex comprises and is selected from following member: polyphosphazene, poly-(vinyl alcohol), polyamide, Merlon, polyalkylene, polyacrylamide, poly alkylene glycol, polyalkylene oxides, polyalkylene terephthalates, polyvinylether, polyvinyl ester, polyvinyl halides, polyvinylpyrrolidone, polyglycolide, polysiloxanes, polyurethane, poly-(methyl methacrylate), poly-(ethyl methacrylate), poly-(butyl methacrylate), poly-(isobutyl methacrylate), poly-(N-Hexyl methacrylate), poly-(isodecyl methacrylate), poly-(lauryl methacrylate), poly-(phenyl methacrylate), poly-(acrylic acid methyl ester .), poly-(isopropyl acrylate), poly-(Isobutyl 2-propenoate), poly-(octadecyl acrylate), polyethylene, polypropylene, poly-(ethylene glycol), poly-(oxirane), poly-(PETP), poly-(vinyl acetate), polrvinyl chloride, polystyrene, polyvinylpyrrolidone, Pluronic, polyvinyl phenol, saccharide (glucosan for example, amylose, hyaluronic acid, poly-(sialic acid), heparan, heparin, or the like); Poly-(aminoacid), for example poly-(aspartic acid) and poly-(glutamic acid); Nucleic acid and copolymer thereof.In another exemplary embodiment, junctional complex comprises and is selected from following member: Polyethylene Glycol (＂ PEG ＂) and polypropylene glycol (＂ PPG ＂) or their mixture.In another embodiment, described PEG or PPG comprise many monomer subunits, and its number is the integer from about 1-about 5000.In another exemplary embodiment, described many monomer subunit numbers are integers of from about 10 to about 1000.In another exemplary embodiment, described many monomer subunit numbers are integers of from about 10 to about 500.In another exemplary embodiment, described many monomer subunit numbers are integers of from about 20 to about 400.In another exemplary embodiment, described many monomer subunit numbers are integers of from about 20 to about 250.In another exemplary embodiment, described many monomer subunit numbers are integers of from about 50 to about 200.In another exemplary embodiment, described many monomer subunit numbers are integers of from about 50 to about 125.In another exemplary embodiment, described many monomer subunit numbers are integers of from about 50 to about 100.In another exemplary embodiment, described many monomer subunit numbers are integers of from about 60 to about 90.In another exemplary embodiment, described many monomer subunit numbers are integers of from about 60 to about 90.In another exemplary embodiment, described junctional complex is selected from diaminourea poly-(ethylene glycol), poly-(ethylene glycol) and combination thereof.For not with heparin-bounding biomolecule, it also is feasible directly being conjugated to polymer support or being connected by junctional complex (for example PEG, amino PEG and diaminourea PEG).
I.a) anti-platelet agents
[0217] in an exemplary, described biomolecule is an anti-platelet agents.The non-limiting example that can be used for the anti-platelet agents of the present composition comprises adenosine diphosphate (ADP) antagonist or P
2Y
12Antagonist, phosphodiesterase (PDE) inhibitor, adenosine reuptake inhibitor, the vitamin K antagonist, heparin, hyparinoids from animal organs, direct thrombin inhibitor, glycoprotein I IB/III inhibitor, the NSAID of aspirin, non-aspirin, antithrombase, and officinal salt, isomer, enantiomer, comprise the polymorph of amorphous form, solvate, hydrate, cocrystallization, complex, active metabolite, reactive derivative and trim, its prodrug etc.
[0218] ADP antagonist or P
2Y
12Adp receptor on the antagonist blocking platelet cell membrane.This P2Y12 receptor platelet aggregation, by fibrinous hematoblastic be important aspect crosslinked.The blocking-up of this receptor has suppressed hematoblastic gathering by the activation of blocking-up glycoprotein iib/iiia approach.In another exemplary embodiment, anti-platelet agents is ADP antagonist or P
2Y
12Antagonist.In another exemplary embodiment, anti-platelet agents is a thienopyridine.In another exemplary, ADP antagonist or P
2Y
12Antagonist is a thienopyridine.
[0219] in another exemplary, ADP antagonist or P
2Y
12Antagonist is to be selected from following member: sulfinpyrazone, ticlopidine, clopidogrel, prasugrel, R-99224 (active metabolite of the prasugrel that sells by Sankyo), R-1381727, R-125690 (Lilly), C-1330-7, C-50547 (MillenniumPharmaceuticals), INS-48821, INS-48824, INS-446056, INS-46060, INS-49162, INS-49266, INS-50589 (InspirePharmaceuticals) and Sch-572423 (Schering Plough).In another exemplary, ADP antagonist or P
2Y
12Antagonist is ticlopidine hydrochlorate (TICLID
TM).In another exemplary, ADP antagonist or P
2Y
12Antagonist is to be selected from following member: sulfinpyrazone, ticlopidine, AZD6140, clopidogrel, prasugrel and composition thereof.In another exemplary, ADP antagonist or P
2Y
12Antagonist is a clopidogrel.In another exemplary, ADP antagonist or P
2Y
12Antagonist is to be selected from following member: clopidogrel bisulfate (PLAVIX
TM), clopidogrel hydrogenesulphate, clopidogrel hydrobromate, the clopidogrel mesylate, cangrelor tetrasodium salt (AR-09931MX), ARL67085, AR-C66096AR-C 126532, and AZD-6140 (AstraZeneca).In another exemplary, ADP antagonist or P
2Y
12Antagonist is prasugrel.In another exemplary, ADP antagonist or P
2Y
12Antagonist is to be selected from following member: clopidogrel, ticlopidine, sulfinpyrazone, AZD6140, prasugrel and composition thereof.
[0220] the PDE inhibitor is one or more in five hypotypes of blocking-up phosphodiesterase (PDE), prevents in the cell second message,second messenger-adenosine cyclophosphate (cAMP) and encircles Guanosine 5'-Monophosphate (cGMP) by the medicine of various PDE hypotype deactivation.In an exemplary, anti-platelet agents is the PDE inhibitor.In an exemplary, anti-platelet agents is a cAMPPDE inhibitor optionally.In an exemplary, the PDE inhibitor is cilostazol (Pletal
TM).
[0221] the adenosine reuptake inhibitor prevents that cell reuptake adenosine from entering platelet, erythrocyte and endotheliocyte, causes extracellular adenosine concentration to increase.These chemical compound anticoagulant also cause vasodilation.In an exemplary, anti-platelet agents is the adenosine reuptake inhibitor.In an exemplary, the adenosine reuptake inhibitor is persantin (Persantine
TM).
[0222] the vitamin K inhibitor is applied to people and is used for preventing thrombosis (unsuitable grumeleuse in the blood vessel).This people that prevention has deep venous thrombosis, pulmonary infarction, myocardial infarction and apoplexy to be inclined to for primary and secondary is useful.In an exemplary, anti-platelet agents is the vitamin K inhibitor.In an exemplary, the vitamin K inhibitor is to be selected from following member: acenocoumarol, Clorindione, dicoumarol (Dicoumarol), diphenadione, biscoumacetate, phenprocoumon, phenindione, tioclomarol and warfarin.
[0223] heparin is a biological substance, prepares from pig small intestine sometimes.It works by activating Antithrombin III, the Blood clotting of Antithrombin III blocking-up thrombin.In an exemplary, anti-platelet agents is the prodrug of heparin or heparin.In an exemplary, anti-platelet agents is the prodrug of hyparinoids from animal organs or hyparinoids from animal organs.In an exemplary, hyparinoids from animal organs is to be selected from following member: Antithrombin III, and shellfish rice heparin, Fragmin, danaparoid, Yi Nuo heparin, sulphur reach the liver last of the ten Heavenly stems (subcutaneous), edegliparin., OP149CA, Clivarin is wished and booth holder heparin Shu Luo.
[0224] directly thrombin inhibitor (DTIs) is a class medicine of bringing into play anticoagulant effect (delay blood coagulation) by direct Trombin inhibiting.In an exemplary, anti-platelet agents is DTI.In another exemplary, DTI is monovalent.In another exemplary, DTI is a bivalence.In an exemplary, DTI is selected from following member: hirudin, bivalirudin (IV), lepirudin 023 ludon, desirudin, argatroban (argatrob) (IV), dabigatran, dabigatran ester (oral formulations), melagatran, Xi Meijia group, its prodrug and analog.
[0225] the glycoprotein iib/iiia inhibitor works by the GpIIb/III receptor that suppresses on the platelet surface, thereby prevents platelet aggregation and thrombosis.In an exemplary, anti-platelet agents is the glycoprotein iib/iiia inhibitor.In an exemplary, the glycoprotein iib/iiia inhibitor is to be selected from following member: abciximab, eptifibatide, tirofib and prodrug thereof.Because these medicines only can pass through intravenous administration, so the prodrug of glycoprotein iib/iiia inhibitor is useful for oral administration.
[0226] antithrombase also can be used for the present invention.In an exemplary, anti-platelet agents is the antithrombase that is suitable for the oral administration form.In another exemplary, antithrombase is to be selected from following member: alteplase, Ahylysantinfarctase, treplase, brinase, Drotrecoginalfa, plasmin, PROTEIN C, reteplase, Saruplase, streptokinase, tenecteplase, urokinase.
[0227] in an exemplary, anti-platelet agents is to be selected from following member: aloxiprin, Beraprost, caicium acetylsalicylate carbamide, cloricromen, defibrotide, Abbott-36683, epoprostenol, indobufen, iloprost, G-137, rivaroxab (oral FXa inhibitor) treprostinil, trifluoro vinegar sulphuric acid, or its prodrug.
Connect biomolecule and/or cell method to biomimetic scaffolds
[0228] can biomolecule and/or cell be connected on the biomimetic scaffolds with several different methods.In one embodiment, biomolecule can non-covalent embedding or is absorbed on the first cellulosic polymers support as herein described.
[0229] in another exemplary, biomolecule is direct or covalently bound to the first cellulosic polymers support by junctional complex.Covalently bound be by the complementary interaction group on the first fibroid support and biomolecule or cell between react and form, or form, or reaction formation between the junctional complex on the first fibroid support and biomolecule or the cell by reaction between the junctional complex on the first fibroid support and biomolecule or the cell.
[0230] being used to implement complementary interaction functional group of the present invention and response type is that the technical staff of bioconjugates chemical field is known.The favourable response type that can be used for reactive functional groups of the present invention is those that react under gentle relatively condition at present.These include but not limited to nucleophilic displacement of fluorine (for example reaction of amine and alcohol and carboxylic acid halides, active ester), electrophilic substitution (for example enamine reaction) and adding carbon-to-carbon and carbon-hetero atom multikey (for example Michael reaction, Diels-Alder additive reaction).These and other useful reactions are described in for example March, Advanced Organic Chemistry, the third edition, John Wiley ﹠amp; Sons, New York, 1985; Hermanson, Bioconjugate Techniques, Academic Press, San Diego, 1996; With Feeney etc., Modification of Proteins; Advances in Chemistry Series, Vol.198，AmericChemical?Society，Washington，D。C.，1982。
[0231] useful reactive functional groups comprises, for example:
(a) carboxyl and various derivant thereof include but not limited to the N-hydroxy-succinamide ester, N-hydroxybenzotriazole ester, and acid halogen, acylimidazole, thioesters, right-the nitrobenzophenone ester, alkyl, alkenyl, alkynyl and aromatic ester;
(b) oh group, it can change into ester, ether, aldehyde etc.
(c) haloalkyl, wherein halogen can after by nucleophilic group for example, amine, the carboxylate anion, the mercaptan anion, carbonium anion, or alkoxide ion replaces, thereby produce the covalently bound of new group in the site of halogen atom;
(d) diene affinity thing group, it can participate in the Diels-Alder reaction, such as maleimide base group;
(e) aldehydes or ketones group, deriving after making becomes possibility, and it passes through carbonyl derivative, for example formation of imines, hydrazone, semicarbazones or oxime, or by realizing such as Grignard additive reaction or lithium alkylide additive reaction mechanism;
(f) after sulfonyl halide groups is used for the reaction of amine, for example in order to form sulfa drugs;
(g) thiol group, it can be converted into disulphide or react with carboxylic acid halides;
(h) amine or mercapto groups, it can be by for example acidylate, alkylation or oxidation;
(i) alkene, it can carry out for example cycloaddition, acidylate, Michael additive reaction etc.;
(j) epoxide, it can react with for example amine or hydroxy compounds; With
(k) phosphoramidite is used for the synthetic standard of nucleic acid functional group with other.
[0232] in an exemplary, reactive functional groups is selected from as follows:
R wherein
31And R
32Be independently selected from following member: replace or unsubstituted alkyl, replace or unsubstituted assorted alkyl, replace or unsubstituted cycloalkyl, replace or unsubstituted Heterocyclylalkyl, replace or unsubstituted aryl, replace or unsubstituted heteroaryl.
[0233] the other example of reactive functional groups, and the corresponding functional group that can react with it is provided in the following table:
Table 1
Possible reactive substituents and the site of reacting therewith
The functional group of reactive functional groups correspondence
The succinimide ester primary amine, secondary amine, hydroxyl
The anhydrides primary amine, secondary amine, hydroxyl
The acyl azide primary amine, secondary amine
Isothiocyanate, isocyanates amino, mercaptan, hydroxyl
Sulfonic acid chloride amino, hydroxyl
Sulfuryl fluoride
Hydrazine, aldehyde, ketone
The hydrazine that replaces
Azanol, amino, hydroxyl
The azanol that replaces
Acid halide amino, hydroxyl
Haloacetamide, maleimide mercaptan, imidazoles, hydroxyl, amino
The carbodiimides carboxyl
The phosphoramidite hydroxyl
Azide alkynes
For example, for chemical compound of the present invention being connected to the hydroxylic moiety on the serine, exemplary reactive functional groups comprises succinimide ester, anhydrides, isothiocyanate, sulfocyanic ester, sulfonic acid chloride, sulfuryl fluoride, acid halide, Haloacetamide, maleimide and phosphoramidite.
[0234] can selective response functional group, make them not participate in or disturb and set up the required reaction of The compounds of this invention.Perhaps, can make reactive functional groups not participate in reaction by there being protecting group.Those skilled in the art understand also and know and how to protect special groups, make its not selected setting of disturbance reponse condition.About the example of useful blocking group, for example referring to people such as Greene, Protective Groups in Organic Synthesis, John Wiley﹠amp; Sons, New York, 1991.
[0235] in an exemplary, carboxylic moiety is connected to amino part.First carboxylic moiety and carbodiimides reaction are to generate O-acyl group isourea, and it can be connected two-part amide moieties to produce with amino partial reaction.
[0236] in an exemplary, can realize being connected of biomolecule and cellulosic polymers support as herein described by several method.For example, biomolecule (such as anti-platelet agents) can be connected to the optional polymer support as herein described that comprises linking group, this is by (being that 1-ethyl-3 (3-dimethylamino-propyl carbodiimides/N-hydroxyl sulfo group-butanimide) is handled and to be realized with EDC/ sulfo group-NHS, being connected of the carboxyl of this amino terminal that helps cellulosic polymers and biomolecule, or promote to be connected to be incorporated into cellulosic polymers contain the amine junctional complex.Should be noted that connection can be opposite situation, as (carboxyl can be on cellulosic polymers, and amido can be on biomolecule).Be readily appreciated that as those skilled in the art EDC/ sulfo group-NHS can be replaced by any suitable group, includes but not limited to: N-5-nitrine-2-Nitrobenzol acyloxy butanimide; P-phenylazide acyl group bromide; P-azidophenyl Biformyl; N-4-(azidophenyl sulfo-) phthalimide; Two (sulfosuccinimide base) suberate; Two-the maleimide hexane; Two [2-(butanimide oxygen base ketonic oxygen base)-ethyl] sulfone; 1,5-two fluoro-2,4-dinitro benzene; 4,4 '-two isothiocyanos-2,2 '-the disulfonic acid stibene; Dimethyl-adipimidate-2 hydrochlorate; Heptan two imidic acid dimethyl ester dihydrochloride; Hot two imidic acid dimethyl ester dihydrochlorides; Two mercaptan two (butanimide propionic ester); Disuccinimidyl suberate; Tartaric acid two succinimide esters; 3, the two third imidic acid dimethyl ester dihydrochlorides of 3-dithio; 4,4 '-the two azidomethyl phenyl nitrogenize things of dithio; 3,3-dithio two (sulfosuccinimide base-propionic ester); Ethyl-4-azidophenyl-1,4-dithio-butyl imines ester; Ethylene glycol bis (succinimido-succinate); 1-nitrine-4-fluoro-3-Nitrobenzol; N-hydroxy-succinamide base-4-azidobenzoic acid ester; Methyl-4-azido benzoyl imines ester; M-maleimide benzoyl-N-hydroxyl sulfo group-succinimide ester; N-hydroxy-succinamide base-4-azidosalicylic acid; P-nitrobenzophenone-2-diazonium-3,3,3-three fluoro-propionic esters; N-succinimido (4-azidophenyl)-1,3 '-two-thiopropionate; Sulfosuccinimide base 2-(m-nitrine-o-nitro-benzoyl amino)-ethyl-1,3 '-dithio propionic ester; N-succinimido-6 (4 '-nitrine-2 '-nitro-phenyl-amino) alkyl caproate; Sulfosuccinimide base 2-(p-nitrine salicyl-acyl ammonia) ethyl-1,3 '-dithio-propionic ester; N-succinimido (4-iodo acetyl group) amino-benzoate; 4-(N-maleimide ylmethyl) cyclohexane extraction-1-carboxylic acid succinimide ester; Succinimido 4-(p-dimaleoyl imino phenyl)-butyrate; N-succinimido 3-(2-pyridine dithio) propionic ester; Two [2-(sulfosuccinimide base oxygen base-carbonyl-oxygen base) ethyl] sulfone; Disulfo succinimido tartrate; Ethylene glycol bis (sulfosuccinimide base)-succinate; M-dimaleoyl imino benzoyl-N-hydroxyl sulfo group-succinimide ester; Sulfosuccinimide base (4-azidophenyl dithio)-propionic ester; Sulfosuccinimide base 6-(4 ' nitrine-2 '-nitro-phenyl amino) alkyl caproate; Sulfosuccinimide base (4-iodo acetyl group) amino-benzoate; 4-(N-dimaleoyl imino-methyl) cyclohexane extraction-1-carboxylic acid sulfosuccinimide ester; Sulfosuccinimide base 4-(p-dimaleoyl imino-phenyl) butyrate; With 2-imino group sulfane hydrochlorate.
II.f3) pharmaceutically acceptable excipient/pharmaceutical preparation
[0237] pharmaceutically acceptable excipient also can be included in the compositions with polymer support of the present invention.In an exemplary, the invention provides compositions, said composition is to comprise a) polymer support of the present invention; And b) pharmaceutical preparation of pharmaceutically acceptable excipient.In an exemplary embodiment, this pharmaceutical preparation is the polymer support that wherein has pharmaceutically acceptable excipient.In an exemplary, this pharmaceutically acceptable excipient is selected from as follows: inert diluent, granulating and disintegrating agent, binding agent, releasing agent and time delay material.
[0238] pharmaceutical preparation of the present invention can take to be applicable to the various forms of selected route of administration.One of ordinary skill in the art will recognize that the various synthetic methods that can be used for preparing the nontoxic pharmaceutical preparation of mixing chemical compound described herein.One of ordinary skill in the art will recognize that the multiple nontoxic pharmaceutically acceptable solvent that can be used to prepare the The compounds of this invention solvate, water for example, ethanol, propylene glycol, mineral oil, vegetable oil and dimethyl sulfoxide (DMSO).
[0239] compositions of the present invention can be passed through surgical incision, the part, or parenteral is used with dosage unit and is comprised conventional nontoxic pharmaceutically acceptable carrier, the preparation of adjuvant and excipient.
[0240] in another exemplary, compositions as herein described and/or polymer are the parts of test kit.This test kit can comprise instruction the inventive method and/or describe the service manual of the component purposes of this test kit.
III.
The purposes of compositions
[0241] in yet another aspect, the present composition (for example polymer support) can be used for the experimenter to replace, and regenerates or improves biological function.In an exemplary embodiment, said composition can replace, and regenerates or improves the function of experimenter's nerve or function or the function of skin or the function of blood vessel of muscle.In yet another aspect, the invention provides the method for treatment experimenter damage, described method comprises: (a) allow described experimenter contact with the present composition of the medicine effective quantity that is enough to treat damage.In an exemplary, said composition contact experimenter's injury site.In another exemplary, described damage is selected from as follows: the nerve of disconnection, the nerve of damaged, the muscle of disconnection, the muscle of damaged, the blood vessel of disconnection, the blood vessel of damaged, the skin of skin wound and contusion.In yet another aspect, the present invention improves the method that the experimenter organizes growth, and described method comprises: (a) allow described experimenter contact with the present composition of the medicine effective quantity of the growth that is enough to promote described tissue.In an exemplary embodiment, this tissue is optional from as follows: muscular tissue, vascular tissue, nervous tissue and skin histology.Said composition can be used to test its effectiveness in external or body.In another exemplary embodiment, the experimenter is an animal.In another exemplary, this animal is selected from as follows: people, Canis familiaris L., cat, horse, rat and mice.
[0242] following is the example of present composition purposes.
III.a) relate to neural purposes
[0243] in an exemplary, compositions as herein described can be used for replacing nerve disconnection or damage.A purposes is to be used to damage peripheroneural regeneration.Peripheral nerve injury can be by wound, autoimmune disease, and institutes such as diabetes cause.Peripheral nervous is made up of nerve fiber, and this nerve fiber reaches whole body target of various end last by health from spinal cord.Peripheral nerve injury causes motion and sensory function in nerve ending target partial loss at least.The most serious damage type, the neural disconnection fully and the big damage breach of formation between neural residul end near-end and far-end.Can regenerate in the nerve fiber of near-end but can not act on the breach of several millimeters long equally effectively.Therefore will damage breach is urgent with the material bridging that effectively guides nerve fiber from the nearly section of nerve to the nerve fiber regeneration of nerve section far away.
[0244] peripheral nervous be damaged and no longer successive clinical setting under, can use at least a vertically aligned cellulosic polymers support as herein described.This polymer support has pipe, fills the shape of pipe or bar.Cause neural discontinuous damage to usually occur in extremity.This aligned fibrous support can be implanted the answer that these zones are passed the neuranagenesis of damage breach with raising and allowed quadruped locomotion and sensor capability.This support can be used for all situations that relates to the nerve that is interrupted damaged, and wherein the breach between neural residul end enough directly connects preventing greatly again.Under every kind of situation, this support with bridging should nerve two ends, be sewn onto the regeneration of nerve segment and raising and guiding nerve fiber from the nearly section of nerve to nerve section far away.The vertical aligned fiber that constitutes support will be arranged in the essentially identical orientation of nerve fiber on.Therefore, this aligned scaffold fibers can provide the specific clue that instructs, the regeneration that this clue guiding nerve fiber is effectively passed the damage breach.Should vertically arrange the pipe polymer support can promote the guiding growth to work by the initial stage to the nerve fiber that is positioned at proximal end perimeter.This shaft-like and fill tubular vertically aligned polymer support may guide continuously all nerve fibers from near-end to the distal nerve sections.But also load biomolecule of this polymer support is extracellular matrix protein matter for example, polypeptide, and somatomedin and/or differentiation factor are with the regeneration of further raising and guiding nerve fiber.The extracellular matrix protein matter that can be added to polymer support comprises collagen, laminin, and fibronectin.The somatomedin that can be added to polymer support comprises basic fibroblast growth factor, nerve growth factor and vascular endothelial cell growth factor.The polypeptide that can be added to polymer support comprises polylysine, based on polypeptide and the laminin imitation polypeptide of RGD.Other biomolecule that can be added to this polymer support comprise heparin and heparan sulfate proteoglycan.These biomolecule also can be used for non-covalent capture layer Fibronectin, and VEGF and BFGF are to the cellulosic polymers support.
[0245] can and regulate the particular demands of size to this support plastotype with coupling patient's nerve injury.For example, this pipe is filled the internal diameter of pipe and the overall diameter of shaft-like polymer support and be can be about 1mm to about 20mm.In another exemplary, this diameter arrives about 8mm for about 2mm.In another exemplary, this diameter arrives about 10mm for about 5mm.In another exemplary, this diameter arrives about 15mm for about 2mm.In another exemplary, this diameter arrives about 18mm for about 12mm.In another exemplary, this diameter can be about 1mm, 1.5mm, 2mm, 2.5mm, 3mm, 3.5mm, 4mm, 4.5mm, 5mm, 5.5mm, 6mm, 6.5mm, 7mm, 7.5mm, 8mm, 8.5mm, 9mm, 9.5mm, 10mm, 10.5mm, 11mm, 11.5mm, 12mm, 12.5mm, 13mm, 13.5mm, 14mm, 14.5mm, 15mm, 15.5mm, 16mm, 16.5mm, 17mm, 17.5mm, 18mm, 18.5mm, 19mm, 19.5mm, 20mm, the specific dimensions that 21mm is complementary as the nerve with damaged.Stent length can be at 1cm to changing to adapt to large-scale damage breach between about 50cm.In another exemplary, stent length arrives about 15cm for about 4cm.In another exemplary, stent length arrives about 30cm for about 14cm.In another exemplary, stent length arrives about 5cm for about 1cm.In another exemplary, stent length arrives about 8cm for about 2cm.In another exemplary, stent length can be about 1cm, 1.5cm, 2cm, 2.5cm, 3cm, 3.5cm, 4cm, 4.5cm, 5cm, 5.5cm, 6cm, 6.5cm, 7cm, 7.5cm, 8cm, 8.5cm, 9cm, 9.5cm, 10cm, 10.5cm, 11cm, 11.5cm, 12cm, 12.5cm, 13cm, 13.5cm, 14cm, 14.5cm, 15cm, 15.5cm, 16cm, 16.5cm, 17cm, 17.5cm, 18cm, 18.5cm, 19cm, 19.5cm, 20cm, 20.5cm.The vertical aligned fibrous support of form of ownership can serve as the substituent of neural autograft, and it is the most widely used now but is far from the ideal form for the treatment of nerve injury.This support also can be used for bridging by the outer long damage breach of current synthetic neural guide product coverage.For example, can be surpassed 3cm by the damage breach of bridging.In an exemplary, the experimenter has long damage breach, and shaft-like polymer support or filling pipe polymer support can be the most preferred support shape that is used to pass long damage breach neuranagenesis.
[0246] under the clinical setting, wherein peripheral nervous be damage but not disconnect, vertically aligned fibrous support of the present invention can be made into to be shaped as thin slice and as twining neural coating and/or can being made into to be shaped as shaft-like or filling tubulose or tubulose and directly being inserted in the damage field.The vertically aligned also available similar biomolecule load as herein described of fibrous support.
[0247] another purposes of support of the present invention is the regeneration that is used for Spinal Cord.Aligned cellulosic polymers support can insert in the damage field with bridging myeloid tissue.This aligned cellulosic polymers support can improve the regeneration with the guide ridges nerve fiber.Also available biomolecule of this support and/or cell loading.Biomolecule can comprise the extracellular matrix protein matter of as above listing, polypeptide, somatomedin and/or differentiation factor and can cause and strengthen the regeneration of spinal nerves.Cell can comprise neural stem cell, neurogliocyte, and/or neural progenitor cell.This cell can replace loss neuron and neurogliocyte and/or can support and strengthen the growth of spinal nerves.
[0248] in another exemplary, vertically aligned polymer pipe support passes the neuranagenesis of damage breach with promotion as neural guiding tube.
III.b) relate to the purposes of skin
[0249] polymer support of the present invention's description can be used for the treatment and the soft tissue regeneration of clinical and individual wound.In one aspect of the invention, the polymer support thin slice is as the graft of damage dressing or outer skin wound.In clinical device, these thin slices can be used for treatment because wound, burn, and ulcer, scratch is scratched, operation, or the wound that causes of other damages.The surgeon can use these to transplant covering and the protection injured area, with the skin tissue of interim replacement loss or damage and the generation of guiding new organization and wound healing to damage field.In clinical device, the micrometer/nanometer sheets of fibres can use the stitching agent, binding agent, or overlapping binder is fixed to wound area.These micrometer/nanometer fiber wound dressings can be cut with the coupling wound size, but or imbrication at edge of wound.
[0250] in another aspect of the present invention, this polymer support thin slice can be made and be used for individual/house and take care of, and this makes the polymer support binder and reach by thin slice is combined with adhesive backing.This stick portion remains on the polymer support thin slice on the position of injured area, and removes can be at fiber degradation or with this tissue welding the time.Also available liquid of this polymer support thin slice or gel adhesive are fixed.
[0251] another aspect of the present invention, big polymer support thin slice can be used as gauze to absorb liquid and the big wound of protection.This polymer support gauze can be wrapped in around the injured area or with adhesive tape and fix.
[0252] in another aspect of the present invention, the polymer support thin slice can be used for treating for example interior wound of amniotic sac of internal soft tissues wound, the ulcer in gastrointestinal tract or the mucosa, and gums damages or subsides inner surgical incision or biopsy etc.The position of filling or covering the damaged tissue zone also can be sewed up or adhere to this polymer support graft.
[0253] polymer support has many features useful to wound healing.At first, polymer support described herein comprises the nanofiber that has nanoporous and can breathe character.They can prevent that microorganism and infectiousness particle from passing, but it allows the air-flow of wound normal healing key and the infiltration of moisture.
[0254] second, the fiber among the present invention is biodegradable, and this allows interim wound covering before the final inwardly growth of new organization.The selection that can determine to be used for the polymer support wound dressing materials with the natural tissues characteristic matching that comprises mechanical strength and degraded/tissue regeneration speed.
[0255] the 3rd, polymer support can combine by embedding or with the various factors, and wherein this factor discharges when degraded.These factors can comprise, but be not limited to epidermal growth factor (EGF), platelet derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transform growth because of in-β (TGF-β), with the tissue depressant (TIMP) of metalloproteases, it has been proved to be in wound healing is useful.Fu，X。et?al.，Wound?RepairRegen，13(2)：122-30(2005)。Other wound healing factor are antibiotic for example, bactericide, and antifungal, argentiferous reagent, analgesic and release oxidation nitrogen compound also can be incorporated in polymer support wound dressing or the graft.
[0256] the 4th, the polymer support graft that is used for wound healing can be used for tissue regeneration faster and more natural organizational structure with cell inoculation.These cells include, but are not limited to fibroblast, keratinocyte, epithelial cell, endotheliocyte, mescenchymal stem cell, and/or embryonic stem cell.
[0257] the 5th, the nanoscale structure of nanofiber polymer support is the structure of the extracellular matrix (ECM) of the many common soft tissues of imitation closely.For example, the nanoscale fiber structurally is similar to the collagen fiber of finding in skin and hetero-organization thereof.This structure can be by being provided for cell migration prevents scar to the organized support of wound formation.This aspect of the present invention, the arrangement of polymer support (opposite with the fiber of random orientation) is arranged the maintenance cell and is important in a organized way, rather than allows it as random alignment in the formation of scar tissue.Aligned polymer support can be orientated to allow to organize faster the inwardly covering of growth and wound at the given axle of wound.
[0258] polymer support is arranged the structure that also will be used for closely mating natural tissues ECM.This can be included in single direction and arrange, and the orthogonal direction decussation is arranged, or the fiber of more complicated fibrous structure.Under situation of the present invention, polymer support is included in the multi-layer fiber that has the special fiber orientation in each layer.Similarly, each polymer support layer also can comprise those that atopen or cell type list before for example.The polymer support generation that this permission can closely be mated the natural tissues structure and be formed.For example, simple polymer support wound dressing or graft can comprise the monolayer alignment aligned fibers.On the other hand, more complicated polymer support skin graft can comprise the eclipsed sheets of fibres of multilamellar decussation, on the base lamina be fibroblast and on the thin slice of top for keratinocyte, and be bFGF on the base lamina and be antimicrobial on the thin slice of top.Other such combinations are possible, depend on patient's specific needs.
III.c) relate to the purposes of vascular system
[0259] polymer support described herein can be used for replacing or walking around various damages, the blood vessel that cuts off or change.In one exemplary embodiment, this pipe or filling pipe polymer support are used for the cononary artery bypass operation.In addition, these grafts by with polymer support blood vessel is replaced fully or by generation as the sheath shell of cover be used for supporting and stable vascular aneurysms (promptly, abdominal aortic aneurysm needs synthetic polymer to replace graft, for example ePTFE or Dacron usually).Other enhancement techniques comprise twines the polymer support thin slice around the aneurysm site.Its purposes is not limited to low body blood vessel and replaces, but can comprise other aneurysmal public sites; For example-Willis around, relate to any local intra-arterial, comprise internal carotid artery, rear quarters is linked up, hindbrain etc.
[0260] in an exemplary, should be used for by the polymer support that sleeve pipe centers on replacing or regeneration vessel.Sleeve pipe can be centered on polymer support to improve its mechanical strength, rigidity, plasticity or any other physics or chemical property.This sleeve pipe can be positioned at around the nanofiber polymer support pipe, for example makes basic nanofiber have special orientation and this sleeve pipe can have identical or different orientation.Non-micron or nanofiber aligned or the random alignment alignment also can serve as shell material or basic nanofiber structure.A plurality of sleeve pipes can be used for generating the multi-ply construction with different physics or chemical property.
Many cells type graft
[0261] tubular structure can be by making the different cell type of zones of different inoculation of polymer support.For example, the cell type 3 that Figure 40 shows cell type 1 with pair cell chamber lining and inoculation encases graft at the cell type 2 at the middle part of graft and on by another zone at graft.
The modification of nano-fiber tubular structure
[0262] in an exemplary, the invention provides the polymer support that is used for vascular system, it does not have biochemistry or cell modification before implantation.In another exemplary, the present invention further comprises poly-(ethylene glycol) or unstained to generate, the not thrombosed brush layer of phase similar biochemical modification, and this layer prevents that platelet and nanofiber from adhering to.But this brush layer covalency is transplanted and be used to reduce thrombosis on the nanofiber polymer support.In another exemplary, polymer support further comprises heparin, hirudin or its combination.Heparin can be in conjunction with Antithrombin III, this thrombin III can be in blood BF Xa and thrombin.Hirudin is the inhibitor of thrombin.Heparin and hirudin can be transplanted on the polymer support by using poly-(ethylene glycol) junctional complex covalency of diaminourea.In an exemplary embodiment, polymer support comprises the PLL fiber.The polymer support of the present invention that can be used for being communicated with experimenter's vascular system has reduced thrombosis and increased to transplant to be renderd a service.
[0263] in another exemplary, the polymer support that is used for getting in touch with vascular system further comprises people's bone marrow derived mescenchymal stem cell.In an exemplary, this mescenchymal stem cell will inoculation at least 24 hours before implantation.In another exemplary embodiment, cell will be inoculated on the graft in implantation in preceding 2 days.
[0264] takes off cell-people's bone marrow derived mescenchymal stem cell or any cell type and will inoculate 2 days as mentioned above.Several hrs before implanting kills this cell, keeps the complete of cellularity and cell surface simultaneously.
[0265] in another exemplary, the polymer support that is used for getting in touch with vascular system further comprises endothelial precursor cell.In an exemplary embodiment, this endothelial precursor cell will be inoculated on the graft in implantation in preceding 2 days.These endotheliocytes can prevent platelet adhesion/activation by efficient surface of cell membrane, and thrombosis and fibrosis form, and this surface of cell membrane comprises heparan sulfate proteoglycan and several by the excretory factor of cell self.
[0266] in another exemplary, the polymer support that is used for getting in touch with vascular system further comprises the human embryo stem cell blood vessel CFU-GM of deriving.These embryonic stem cells can be divided into the blood vessel progenitor cell line and can fully be divided into endothelium and smooth muscle cell under factors stimulated growth by collagen iv integrin signal.
III.d) relate to the purposes of muscle
[0267] the muscle graft treatability ground that comprises polymer support of the present invention implants that intramuscular is used to strengthen because the anathrepsis of important muscle loss after the acute injury.Somatomedin and other biological molecule can be introduced muscle graft to stimulate muscle cell multiplication and differentiation and vascularization, and it will promote the healing of all muscle.Somatomedin can comprise insulin-like growth factor-i (IGF-1), alkaline somatomedin (bFGF), vascular endothelial cell growth factor (VEGF), and platelet derived growth factor (PDGF).This somatomedin can be incorporated into the transplanting species by covalent bond or physiology coating.Relevant method comprises that genetically modified cell is to cross the proteinic like this gene of expression.
[0268] to use be the genetic modification muscular dystrophy for example that is used to have the animal or human of muscle disease in another treatment of graft, it is characterized by the genetic mutation of specific gene.Use for such gene therapy, the sarcoplast with normal gene can be delivered to muscle in graft.Perhaps, this gene can directly be connected on the support with the plasmid DNA form.Transplanting at the body interior support can cause genetic modification to help the organizational development of normal muscle function.Concerning gene therapy purpose, this polymer support is provided for implanting the substrate of the survival of cell and growth and/or serves as the release that is used for plasmid DNA and with the delivery media of post-absorption.
[0269] this graft can be the shape of sheet or pipe.In both cases, machine direction is parallel to the direction of muscle.For Rodents, the scope of the physical size of this support is 0.5-5.0 * 0.5-5.0 * 0.1-0.5cm.For people's graft, the scope of this stent size is 1.0-50.0 * 1.0-50.0 * 0.1-5.0cm.
[0270] muscle that is suitable for this class therapy most be have arrange geometry and near vascular system to nourish those of graft.This muscle comprises biceps, musculus quadriceps, anterior tibia flesh, and gastrocnemius.
[0271] in an exemplary, the pipe of imbedding with sarcoplast can be used for the skeletal muscle of growing.In another exemplary, the pipe of imbedding with mescenchymal stem cell can be used as blood vessel graft.In another exemplary, can be used as nerve graft with the pipe of neural stem cell embedding.In another exemplary, the invention provides the method for making said composition, this method comprises (i) with this polymer and cell inoculation; (ii) with product roll compacting around mandrel, thereby be formed for the tubulose of polymer; (iii) remove this mandrel.In another exemplary, the invention provides the second portion that (iv) first of this polymer is connected to this polymer.This connection is by means of annealing (heat), and bonding (biological example binding agent) or suture are sewed up and realized.
III.e) has the muscle graft of micro-patterning polymer support
[0272] the muscle graft application class with micro-patterning polymer support is similar to the application of aforesaid nano-bracket, except the micro-patterning polymer support is a lamella shape.
[0273] the present invention is by following embodiment example.This embodiment is not intended to limit or limit the scope of the invention.
Embodiment
Myoblastic preparation
[0274] Mus C2C12 sarcoplast (ATCC, Manassas VA) are used to study groups of cells structure and assembling.This sarcoplast is cultivated in growth medium, and this growth medium is by dulbecco ' s Modified Eagle ' s culture fluid (DMEM), and 10% hyclone and 1% penicillin/streptomycin are formed.In order to cause the sarcoplast differentiation and to converge, after sample converged 24 hours, this growth medium was replaced by differentiation culture liquid, and this division culture medium is by DMEM, and 5% horse serum and 1% penicillin/streptomycin are formed.In all experiments, time point was represented by the incubation time in differentiation culture liquid.
The preparation of PLLA nano fiber scaffold
[0275] with biodegradable poly-(L-lactide) (PLLA) (Lactel AbsorbablePolymers, Pelham, AL, 1.09dL/g intrinsic viscosity) be used for making nano fiber scaffold by electrostatic spinning.(Zong，X.，Biomacromolecules，4(2)：416-23(2003))。In brief, this PLLA solution (10%w/v is in chloroform) is delivered to the electrode outlet hole by programmable pump with the flow velocity of 25 ml/min.(GlassmHigh Voltage Inc., High Bridge NJ) are used to apply 20 kilovolts voltage to high voltage power supply.Collecting board is on rotary drum, and this rotary drum is by motor ground connection and control.In order to arrange nanofiber, the support of electrostatic spinning unidirectionally extends to 200% engineering strain at 60 ℃.It is thick that nano fiber scaffold is approximately 150 μ m.This nano fiber scaffold surface is coated with 2% gelatin or fibronectin (5 μ g/cm before cell inoculation
2).Not not being coated with interlayer at gelatin and fibronectin detects at the significant difference aspect cell adhesion and the form.The support of random orientation is with comparing.
[0276] scanning electron microscopy (SEM) is used for the nanofiber arrangement of developing after uniaxial tension.The SEM pictorial display causes arranging the uniaxial tension (Figure 24 A-B) of nanofiber.Average nanofiber diameter in this support is about 500nm, and has the average notch size of about 4 μ m.
Myoblastic growth/sign on the nano fiber scaffold under the differentiation culture liquid
[0277] will merge into the myocyte and in differentiation culture liquid, be cultured to 7 days on the nano fiber scaffold.In order to be determined at cell tissue and the cytoskeletal structure on random orientation and the aligned nano fiber scaffold, to F-actin, myoglobulin heavy chain (MHC) and nucleus carry out fluorescence staining (Supporting Information).F-actin uses the phalloidin of fluorescein (FITC)-put together, and (Molecular Probes, Eugene OR) dyes.(Sigma, St.Louis MO) carry out immunostaining to MHC with the quick MHC antibody of mouse anti skeletal muscle.Nucleus dyes with ToPro dyestuff (Molecular Probes).Use Nikon TE300 microscope and LeicTCS SL Laser Scanning Confocal Microscope to carry out fluorescence microscopy.
Embodiment 4
Little manufacturing of micro-patterning base material
[0278] by improved Thakar, R.G., Biochem Biophys Res Commun, 307 (4): the method among the 883-90 (2003) is made by polydimethylsiloxane (PDMS) or micro-patterning thin film that poly-L-lactide-copolymerization-Acetic acid, hydroxy-, bimol. cyclic ester-copolymerization-6-caprolactone (PLGC) polymer is formed by silicon wafer antitemplate (silicon waferinverse template).In order to generate the micro-patterning template, wafer cleans in acetone, and then cleans with the wash solution (Piranhacid) of sulphuric acid and hydrogen peroxide 4:1.Dry this wafer also applies hexamethyldisiloxane (HMDS) 5 minutes to improve the adhesion of photoresist to base material.At first produce mask with patterning emulsion bar.Parallel strip is that 10-μ m is wide, and 10-μ m interval and 4-cm are long.For with this design transfer to silicon wafer, I-line (OCGOiR 897-10i, ArchChemicals, Norwalk, CT) speed of photoresist 820RPM rotates to wafer last 1 minute, generates the thick photoresist layer of about 2.8-μ m.This photoresist is exposed to before the ultraviolet with KS aligner then, this wafer cures 60 minutes with this photoresist that hardens at 90 ℃.Carry out post exposure bake 60 seconds to help the photoresist typing and to order about the diffusion of this photoproduct at 120 ℃.Use the development of photoresist 60 seconds of 4262 pairs of exposures of development of photoresist liquid OPD, the wafer with unexposed photoresist is put into primer baking oven 15 minutes to allow remaining photoresist typing at 120 ℃.At this point, wafer can be used for the preparation of PDMS thin film at any time.The template that is used for the PLCG thin film fabrication need utilize STS Deep Reactive Ion Etcher etched additional step 2-3 minute to generate the dark microgroove of about 2 μ m.
[0279] the PDMS thin film of micro-patterning and non-pattern according to producer (Sylgard 184, Dow Corning, guidance MI) prepares.After the degassing, 15gPDMS is poured over wafer last under vacuum.Wafer with PDMS is placed on the speed rotation 30 seconds of photoresist rotator and 100rpm, and with the speed rotation of 200rpm 2 minutes, it formed the PDMS thin film with about 350 μ m equal thickness then.Wafer with spin coated PDMS was preserved at room temperature 10 minutes, was placed on 80 ℃ the baking oven polymerization with permission PDMS in interior 15 minutes then.After curing, the PDMS cool to room temperature is removed from wafer then.After manufacture process, this PDMS film carries out supersound process and cleans in water.In order to sterilize and to promote cell adhesion, this film applied 2% gelatin 30 minutes with oxygen plasma treatment and before cell inoculation.
[0280] for biodegradable micro-patterning thin film, with 70:10:20 component ratio (M
nAbout 100,000) use three blocks copolymer p LGC (Aldrich, St.Louis, MO).PLGC solution prepares concentration in chloroform is 50mg/mL and stirs in bench mixer (stirplate) up to dissolving.This solution is poured on the silicon mold and makes solvent evaporation then, forms thin polymer film.After manufacturing, the PLGC thin film is in the sterilization of 70% ethanol 2 hours and rinsing in PBS.Before cell inoculation, this thin film applies 30 minutes to strengthen the connection of cell with 2% gelatin.
Embodiment 5
(SEM) characterizes compositions by scanning electron microscopy
[0281] compositions or the sample that comprises cell fixed to carry out SEM by 2% glutaraldehyde in being dissolved in 0.1M sodium cacodylate buffer liquid.After with the ethanol continuous dehydration, drying sample and sputter coating iridium and gold: the particle of platinum (40: 60) is 10-15nm to thickness.Sample is developing under environmental scanning electron microscope (Philips XL-30).
Embodiment 6
Immunofluorescence dyeing
[0282] sample was fixed on 4% paraformaldehyde 15 minutes, with 0.5% saturatingization of Triton X-100 10 minutes, and anticipated 30 minutes with 1% bovine serum albumin (BSA).F-actin assembling was dyeed 1 hour by phalloidin (5U/mL, the Molecular Probes) incubation of fluorescein (FITC)-put together.For myoglobulin heavy chain dyeing, the quick myoglobulin heavy chain of sample and mouse anti skeleton (87 μ g/mL, Sigma) incubations, the anti-mice of donkey (6.25 μ g/mL that put together with FITC-then, Jackson Immuno Research, West Grove, PA) antibody incubation together.Nucleus was being used ToPro (1 μ M, MolecularProbes) dyeing in the past with Nikon TE300microscope or LeicTCS SL confocal microscope developing.Confocal images is represented the bidimensional projection of three-dimensional stacked image.
BrdU mixes
[0283] for cell cycle analysis, sample with bromodeoxyribouridine (BrdU, 1:1000, Amersham, Piscataway, NJ) pulse 2 hours in growth medium is fixed on 4% paraformaldehyde then and washs in phosphate buffered saline (PBS) (PBS).This sample was anticipated 30 minutes with 50% methanol, and then 0.5% saturatingization of Triton X-100 10 minutes, the 2N hydrochlorate was handled 30 minutes then.Afterwards, and sample mouse anti-BrdU (2.5 μ g/mL, BDBiosciences, Bedford, MA) incubation is that FITC-puts together the anti-mice of donkey (6.25 μ g/mL) antibody then.With iodate third ingot (1 μ g/mL, Molecular Probes) pair cell nuclear staining.
Embodiment 8
The analysis of cell fusion and propagation
[0284] the painted fluorescence immunoassay image of anti-skeletal muscle globulin is at least 5 representational high-power 40x) and low-power (10x) visual field under obtain.Use SPOT4.0.5 (Diagnostic Instruments, Sterling Heights MI), utilize high x magnification and low x magnification to the myotube width respectively to software, length and quantize with respect to the arrangement of the axle of arranging nanofiber or microgroove.The minimum myotube of 0 degree has been arranged value representation and has been arranged in parallel and the maximums of 90 degree are represented arranged vertical along the nanofiber axle.Arrangement analysis for the PDMS base material of the nano fiber scaffold of random orientation and non-patterning uses and arranges axle arbitrarily.In addition, to fusion nucleus, the percentage ratio of BrdU-positive cell and striated myotube quantizes and is average with high power.Mean breadth is quantized with average.Image series under low x magnification merges with quantitative myotube length.All data are represented with average ± standard deviation (n 〉=3).Carry out multiple comparisons by two groups studentShi t check computational statistics significances or the variance analysis of using Holm ' s to adjust.
Embodiment 9
Pipe
[0285] sarcoplast broke up 7 days on rectangular arranged nanofiber thin slice.In order to generate three dimensional structure, this nanofiber thin slice is around the steel pole roll compacting (Figure 27) of 1-2mm diameter.This tubular structure is sewed up by the 7-0Ticron on these tubular structure two ends and is fixed.This sample low temperature cutting is used for fabric analysis then.This low temperature cutting sample is by the hematoxylin and the eosin (H﹠amp of routine; E) immunofluorescence dyeing of dyeing and F-actin is analyzed.
[0286] the tubular structure diameter of Zhi Zaoing is that about 2-3mm and longitudinal length are 10mm.The H﹠amp that is used for the configuration in these structure cross sections; The tubular structure of this structure of E dyeing proof can successfully be made (Figure 28).Can on the entire bracket layer, see remarkable purple-painted nuclear, although more many cells are visible on the surface of each layer.The confocal microscopy proof Premeabilisation of cells of immunofluorescence dyeing F-actin is to this multi-ply construction interior (Figure 29).Cell adopts extended form and arranges according to the direction of nanofiber.These results have proved and have used nanometer polymer to handle the feasibility of arranging three-dimensional skeletal muscle.
The growth of nanofiber polymer
[0287] biodegradable (L-lactide) (PLLA) (Lactel absorbable polymer that gathers, Pelham, AL, 1.09dL/g intrinsic viscosity) be used for before by Rosen etal., Ann Plast Surg., the described electrostatic spinning of 25:375-87 (1990) is made nano fiber scaffold.PLLA solution (10%w/v is in chloroform) is delivered to ground connection collecting board in high electric field by programmable pump, produces nanofiber at random.In order to arrange nanofiber, the support of this electrostatic spinning is in the engineering strain of 60 ℃ of uniaxial tensions to 200%.It is thick that nano fiber scaffold is approximately 150 μ m.Scanning electron microscopy (SEM) is used for the nanofiber of developing after uniaxial tension and arranges.The SEM pictorial display produces the uniaxial tension (Figure 12 A-B) of highly arranging nanofiber.Average nanofiber diameter is approximately 500nm.
[0288] for the chemical modification nanofiber, a kind of ECM protein (laminin) and a kind of neurotrophic factor (the basic fibroblast factor or bFGF) are chosen as representational example.Laminin and bFGF have demonstrated the extension that promotes external neurite.Manthorpe?et?al.，J?Cell?Biol.，97：1882-90(1983)；Rydel?etal.，J?NeuroscI.，7：3639-53(1987)。In addition, these two kinds of protein link to each other with heparin by their heparin binding domain.Heparin has also demonstrated and has prevented the bFGF degraded and played pivotal role in the bFGF cellular signal transduction pathways.Gospodarowicz?et?al.，JCellPhysiol.，128：475-84(1986)；Sakseletal.，J?CellBiol.，107：743-51(1988)；Yayon?et?al。Cell，64：841-8(1994)。
[0289] the heparin functionalized nano-fiber uses poly-(ethylene glycol) (two-amino-PEG) generate as junctional complex molecule (Figure 12 C) of diaminourea.At first, the density of the pendant carboxylic group on the PLLA nanofiber is by (Sigma, St.Louis MO) handle and increase with 0.01N NaOH.Use zero-length cross-linking agent 1-ethyl 3-(3-dimethylaminopropanecompounds) carbodiimide hydrochloride (EDC) and N-the hydroxysulphosuccinimide ((PierceBiotechnology of sulfo group-NHS) then, Rockford, IL) (MW 3400, and Sigma) molecule is covalently bound with the carboxyl on the PLLA nanofiber with two-amino-PEG.By EDC and sulfo group-NHS that heparin (Sigma) molecule is covalently bound with the free amine on two-amino-PEG molecule then.Any residual activity site on nano fiber scaffold is by blocking at phosphate buffer saline solution (PBS) incubation sample at the 10%w/v glycine.Then bFGF (50ng/cm2, Peprotech, Rocky Hill, NJ) and laminin (10 μ g/cm2 Sigma) combine and are fixed on the surface of nanofiber with heparin to allow them with nano fiber scaffold order incubation in PBS solution.
[0290] the bFGF molecule is verified with the elisa technique of revising by use that is connected of the PLLA nanofiber of heparin functionalization.For the relatively fixing efficient of bFGF different modes, three of bFGF incubations that are used among the PBS have same size, and are untreated, and two-amino-PEG modifies and the nano fiber scaffold of the nano fibrous membrane of heparin functionalization.Be used in the PBS solution all films of 1% bovine serum albumin (BSA) incubation then to minimize the passive absorption of antibody.Subsequently, anti--bFGF mouse monoclonal IgG antibody (R ﹠amp of puting together with HRP-of sample; D Systems, Minneapolis, MN) incubation.Thoroughly this film of washing is transferred to microcentrifugal tube and is used HRP substrate solution (hydrogen peroxide and chromagen, tetramethylbenzidine) incubation.Use 2N sulphuric acid to stop this reaction then.Use absorptance (450nm wavelength) reading of spectrophotometer then to this solution.Being adsorbed on the negative control sample of non-specific bFGF antibody tested, and is considered to inappreciable (data not shown).Result (Figure 12 D) shows that the bFGF coating is greatly effective on the nanofiber with the heparin functionalization.These results further on tissue culturing polystyrene's base material of poly-(acrylic acid) coating check and with two-amino-PEG and heparin to put together (Figure 12 E) in the employed same way as of PLLA nanofiber.
[0291] the DRG tissue of gathering in the crops from the P4-P5 rat is used to study the neurite extension on the nano fiber scaffold.Be supplemented with B27 and 0.5mM L-glutaminate (invitrogen, Carlsbad, CA) DRG that cultivates in the neural basic culture solution on following aligned and non-aligned PLLA nano fiber scaffold organized 6 days: undressed, with laminin (LAM) heparin functionalization and with (LAM+bFGF) of laminin and bFGF heparin functionalization.After 6 days, use immuning fluorescent dyeing analysis neurite extension at In vitro culture.At first specimen in use is fixed with 4% paraformaldehyde, then with 0.5% Triton-X100 saturatingization cell membrane in PBS solution.(SantCruzBiot echnology SantCruz CA) puts together the anti-goat IgG antibody of donkey (Jackson Immunoresearch, West Grove, PA) incubation sample with FITC-subsequently with the polyclonal anti-neurofilament-M of goat (NFM) IgG antibody.This sample is arranged on the microscope slide and uses the Zeiss fluorescence microscope and Leica confocal fluorescence microscopy developing.
[0292] on non-aligned unprocessed nano fiber scaffold, can not observe (Figure 13 is untreated) by the neurite extension of DRG tissue.The neurite extension of organizing from the DRG that cultivates at non-aligned LAM nanofiber is minimum (Figure 13 LAM).Can be observed various neurites extends from the DRG tissue of cultivating at non-aligned LAM+bFGF nanofiber.Neurite is outwards stretching and shortage arrangement concordance from the form of DRG tissue with radius.Be about 1mm from the neurite product of DRG tissue.
[0293] opposite with non-aligned undressed nanofiber, on unprocessed arrangement nanofiber, observe the neurite extension.Neurite organizes two zoness of different to stretch out and arrange along the nanofiber direction from DRG.Figure 14 has shown that neurite stretches out one of from two zones of tissue.Neurite product on undressed nanofiber is about 0.7mm.These results show that aligned nanofiber provides the guidance of inducing from the neurite extension of the interior sensory neuron of DRG tissue.Similar aligned but longer neurite is observed (Figure 14 LAM) on aligned LAM nanofiber, hint ECM protein can further strengthen the guidance of arranging nanofiber.Neurite product on the LAM nanofiber is about 1.3mm.The longest and the most intensive neurite extends in and observes (Figure 14 LAM+bFGF) on the aligned LAM+bFGF nanofiber.The about 4.8mm of neurite product on the LAM+bFGF product.These results are clear have been shown from the chemical clue of laminin and bFGF with from the physics clue of arranging nano fiber basis material, individually or combination, strengthens widely and instructs the extension of neurite from sensory neuron.High power-amplification confocal microscopy is learned the guidance that neurite that proof extends is followed the biologically active nanometer fiber, and is forming different patterns (Figure 15) at random with on the aligned nanofiber.
Embodiment 11
Vertical aligned polymer support pipe
[0294] biodegradable poly-(lactic acid-copolymerization-hydroxyacetic acid) (PLLA) (Lactel absorbable polymer, Pelham, AL, 0.82dL/g intrinsic viscosity) be used for making nano fiber scaffold by electrostatic spinning.PLGA solution (20%w/v is in HFIP) is delivered to the electrode outlet hole with 1 milliliter/hour flow velocity by the programmable pump.High voltage power supply is used to apply 11 kilovolts voltage.The colelctor electrode base material is to connect the ground connection steel core axle that can rotate the motor of mandrel around its major axis.The Teflon adhesive tape is wrapped in a mandrel part on every side to generate the non-conductive area.Mandrel low speed rotation＜15rpm) is electrostatic spinning PLGA fiber simultaneously.Between two parts of separating mandrel by nonconducting Teflon region, replace the deposition that jet produces the PLGA fiber parallel with the mandrel major axis.The even covering of PLGA fiber around mandrel guaranteed in the rotation of mandrel.After finishing electrostatic spinning, the edge of the fibrous pipe of PLGA of electrostatic spinning turns the edge of then with pocket knife and remove this pipe from Teflon band and mandrel.
[0295] arrange for testing fiber, this pipe is along its long axis direction cutting and with optical microscope developing fibre morphology.Most of fibers are vertically being arranged the major axis of pipe (promptly along).
Embodiment 12
Vertical aligned polymer support bar
[0296] biodegradable poly-(lactic acid-copolymerization-hydroxyacetic acid) (PLLA) (Lactel absorbable polymer, Pelham, AL, 0.82dL/g intrinsic viscosity) be used for making nano fiber scaffold by electrostatic spinning.PLGA solution (20%w/v is in HFIP) is to be delivered to the outlet opening of electrode by the programmable pump under 1 milliliter/hour the flow velocity.High voltage power supply is used to apply 11 kilovolts voltage.For the electrostatic spinning process forms shaft-like vertically aligned fiber, can use specific colelctor electrode base material.The colelctor electrode base material can be made up of two grounded metal mandrels that join end to end aligned, wherein air gap is arranged and can place under the outlet opening of electrode (being 15cm) middle (being 2cm).Each mandrel can be connected with electronically controlled motor, and this motor can rotate mandrel in a synchronous manner around the major axis of mandrel.During the process of electrostatic spinning, can the low speed rotation mandrel (＜10rpm) to guarantee the uniform deposition of electrostatic spinning polymer fiber.During the electrostatic spinning process, polymer solution will form to collecting the jet that base material moves.Under these situations, this jet will be passed in the air gap between the end points of two mandrels, form along the aligned fiber of notch length direction (with having the orientation identical with the long axis direction of mandrel).When finishing electrostatic spinning, the polymeric material of this electrostatic spinning can be by using pocket knife and separating with two metal-cored axle head points along the mandrel edge cuts.Form shaft-like electrostatic spinning cellulosic polymers support, it has along the aligned fiber of its long axis direction.
Embodiment 13
Vertically arrange and fill pipe cellulosic polymers support
[0297] in order to form by the vertically vertically aligned polymer fiber filling of aligned fibrous Guan Bingyong, can use following method: at first, the hollow pipe with vertical aligned fiber is an electrostatic spinning.Long in order to make 2cm, internal diameter 0.5cm is aligned filling pipe vertically, can use following condition.At first, vertically aligned hollow pipe can as described hereinly use and have the mandrel electrostatic spinning of the long and diameter of 2cm at least as the non-conductive area of 0.5cm.Vertically aligned then hollow pipe is removed and cut lengths from mandrel.The packing material electrostatic spinning is as highly porous arranged polymeric sheets of fibres as herein described.Aligned nanofiber thin slice can be made into the shape of the appropriate size of hollow pipe lumen.The pipe holder of filling along the aligned polymer fiber of major axis with generation in the aligned then sheets of fibres insertion tube.
[0298] packing material can as aligned fibrous rolled sheet with by vertical the same manufacturing of aligned fibrous bar.To make packing material in order using, can to use methods described herein by aligned fibrous rolled sheet.The aligned cellulosic polymers film of 50 micron thickness can be by the electrostatic spinning manufacturing and can be cut to 2cm x 2cm.Then the mode roll compacting can aligned fiber carried out along rolled sheet length of this thin slice to from one's body.But this thin slice roll compacting is 0.5 centimetre up to the diameter that forms bar.Cut excess stock then.The shaft-like packing material of this roll compacting can insert in the vertically aligned hollow pipe then, utilizes forceps to form vertically aligned filling pipe.In order to make packing material, can use according to methods described herein by vertical aligned fibrous shaft-like cellulosic polymers support.In order to make the bar of appropriate size, two colelctor electrode axle diameters can be 0.5cm and with 2cm at least at interval to generate 2cm air gap at least.After as enforcement scheme 12 electrostatic spinning bars, this bar can use pocket knife to remove, and along the mandrel edge cuts.Rod-shaped scaffold uses forceps to be inserted in the hollow pipe then.
Embodiment 14
The preparation of PLGA nano fiber scaffold
[0299] biodegradable poly-(lactic acid-copolymerization-hydroxyacetic acid) (PLLA) (Lactel absorbable polymer, Pelham, AL, 1.09dL/g intrinsic viscosity) be used for making nano fiber scaffold by electrostatic spinning.PLGA solution (20%w/v is in HFIP) is delivered to the outlet opening of electrode by the programmable pump with 1 milliliter/hour flow velocity.High voltage power supply is used to apply 11 kilovolts voltage.(diameter: 10cm, length: on rotation steel rotary drum 10cm) (referring to Figure 41), this rotary drum ground connection is also passed through step motor control to fiber laydown being coated with aluminium foil.For aligned nanofiber, this colelctor electrode drum is with the speed rotation of 400rpm.For non-aligned fiber, this colelctor electrode drum is with the speed rotation of＜30rpm.It is thick that electrostatic spinning proceeds to the about 130 μ m of the fibrous support of being made up of the 500nm diameter fibers.In order to remove the cellulosic polymers thin slice, twine down polymeric layer and paper tinsel along the length cutting polymer layer of drum and paper tinsel and from drum from this drum.The cellulosic polymers layer is peeled off to make the about 30 long and wide cellulosic polymers thin slices of 10cm from aluminium foil then.
[0300] uses the arrangement of phase contrast microscope (Carl Zeiss) testing fiber and the cellulosic polymers thin slice cut into required size to be fit to given application.For wound healing, the nano fiber scaffold sheet can cut with the size of coupling wound or be expanded to outside the wound.The orientation that can select fiber is to become special angle to arrange with wound to dead axle.
The preparation of the nano fiber scaffold of decussation
[0301] the cellulosic polymers support with cross pattern fiber can form by SOME METHODS.In one embodiment, electrostatic spinning can be by being collected in the fiber on the rotary drum or being used to generate as aligned cellulosic polymers thin slice as described in before this patent by extending non-aligned fiber.These aligned polymer flakes are removed also lamination each other from the colelctor electrode drum then, and the fiber in each thin slice is perpendicular to top and following fiber assortment (Figure 38) simultaneously.In addition, this thin slice can be stitched together for mechanical strength and stability.
[0302] in second embodiment, this decussation pattern can be formed for along the longitudinal fiber electrostatic spinning of drum by utilizing the conductive drum that has non-conducting portion at the center.Diameter is that 10cm and the long steel drum of 10cm can be fixed on the motor.The Teflon adhesive tape can be wide to cover 4cm in drum roll compacting on every side.In order to generate cross pattern, at first this drum will slowly rotate (＜30rpm) pass non-conductive Teflon region along the aligned fiber of the longitudinal axis with generation.Fast rotational is roused (〉 100rpm subsequently) to generate the fiber that aligns with the ground floor arranged vertical.Sedimentary a series of aligned fibrous layers produce cross patterns on the Teflon region thereby in the conversion at a slow speed and between the fast rotational of this drum generation is deposited on.Perhaps, the structure drum, wherein the non-conductive area is to be clipped between two conductive metal regions, this is similar to the design that the present invention describes mandrel before.
[0303] in the 3rd embodiment, the cellulosic polymers support thin slice with the aligned fiber of decussation can use rotary drum as colelctor electrode base material electrostatic spinning.The electrostatic spinning device can outfit described herein be used for the aligned cellulosic polymers thin slice of electrostatic spinning.Aligned fibrous layer is deposited on the rotary drum.This fibrous layer can be peeled off from drum then, revolves to turn 90 degrees and be put back on the bulging colelctor electrode.Then this drum again with at a high speed (＜100rpm) rotation to be to form the deposition of aligned fiber.This process can repeat many times to make the aligned cellulosic polymers support thin slice of decussation, the corresponding fibrous layer arranged vertical that is adjacent of wherein any given fibrous layer.
Embodiment 16
Micrometer/nanometer fiber wound healing
[0304] PLLA micrometer/nanometer sheets of fibres can have arrangement and non-aligned fiber as generation as described in the enforcement scheme 10.Generate in the fibroblast (NHDF) of artificial wound or the monolayer normal human skin of breach defective on following these sheets of fibres: at first, use manual clamp that No. 18 ejector syringe needles are flattened.Secondly, nanofiber PLLA mesh is cut into the size of 1 x 1cm.Syringe needle of flattening and mesh were sterilized 30 minutes under 70% isopropyl alcohol and ultraviolet, and pin fixedly were passed in about the nanofiber-fiber needle on the required orientation of fiber alignment the wound axle to being parallel, and be vertical, or non-aligned (Figure 35).NHDF is combined in following (Figure 35) in Zone Full blend inoculation but this pin prevents cell.Behind bonding cell, remove this pin, " wound " stayed on its position.Culture remains in 37 ℃ of moistening couveuses 1-2 days to allow the time for cell migration and wound covering with 5% carbon dioxide.Before inoculation, NHDF goes into the progress of wound with DiI tracking cell device dyeing (1:2000 dilution factor, 10 minutes) to observe original wound size and monitoring cellular infiltration briefly.
[0305] NHDF on the micrometer/nanometer fiber fixes with 4% paraformaldehyde, changes thoroughly and blocks with 1% bovine serum albumin with 0.1%Triton X-100.For cytoskeleton dyeing, sample was educated 60 minutes with anti-whole actin one temperature resistance, puted together anti-goat IgG two anti-(Jackson ImmunoResearch) incubations 60 minutes with FITC-then.The cytoskeleton feature is used to measure cell orientation and form, and the overall tissue of NHDF monolayer and wound covering degree.NHDF nuclear dyes 5 minutes to allow cell counting with Hoechst.On non-aligned fiber, cell migration and wound covering degree are unique appropriate after 24 hours, but when the long edge-perpendicular of fiber and wound was orientated, it had been strengthened (Figure 36) widely.Also have, NHDF is random orientation on non-aligned fiber, but is orientated with machine direction on aligned fiber.
Embodiment 17
Wound healing
[0306] only use aligned fiber to be used for wound healing as generating PLLA cellulosic polymers support thin slice as described in the enforcement scheme 16.Before cell inoculation, cellulosic polymers support thin slice can be used embodiment 10 described laminins and/or bFGF functionalization, and bFGF is fixed on the described polymer support thin slice and with soluble form and is present in the culture fluid.Generate artificial wound or defective in the monolayer normal human skin fibroblast (NHDF) on embodiment 16 described these thin slices.The fiber alignment of all samples is vertical about the major axis of wound.With embodiment 16 described immunostainings and migration of micrology analysis of cells and wound covering degree.NHDF on the unprocessed fiber did not fully cover wound area after 24 hours, and the NHDF that handles on the fiber (laminin or bFGF) moves quickly and demonstrates enhanced to wound
Wound covering degree (Figure 37).
Embodiment 18
The nanofiber wound healing of animal
[0307] arrange biodegradable polymer nanofiber thin slice and use embodiment 14 described rotary drum colelctor electrodes to generate, these thin slices will be as wound dressing to help the intravital skin of animal tissue repairing.The surgeon will be in the rat back cutting with the thick breach of monoblock.The micrometer/nanometer sheets of fibres is cut into the size of wound, and be sewn onto in the ectoderm to help fibroblast to move to breach.Digital photography is used in wound healing and tissue regeneration, histology and immunohistochemistry monitoring.
Embodiment 19
The method for preparing the hirudin biomimetic scaffolds
[0308] can make and prepare hirudin biomimetic scaffolds of the present invention with the following method:
With poly-(the L-lactic acid) of 15-25 weight/volume percent, poly-(glycolic) but, the polymer or the admixture of its both combination or any other biodegradable/bio-absorbable be dissolved in the solvent (1,1,1,3,3,3-hexafluoro-2-propanol).
2. the salt (sodium chloride, sodium acetate etc.) of 0.1-5 w/w percentage ratio can be joined in the described polymer solution, with the electric conductivity that increases solution and reduce fibre diameter.
3. use electrostatic spinning technology described herein that polymer solution is applied to (axle diameter 0.1-10cm collects dull and stereotyped) on the substrate.
4. make the interior solvent evaporation of electrostatic spinning pipe several hours.
With described pipe in 70% ethanol and be placed under the UV sterilization conditions and sterilized at least 30 minutes.
6. with the described sample of distilled water cleaning down.
7. handled described pipe at least 10 minutes with 0.01-1 standard NaOH, to increase the carboxyl quantity on the polymer.
8. with the described sample of distilled water cleaning down.
9. with 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC or EDAC) and N-hydroxysulphosuccinimide (Sulfo-NHS) be connected to functionality amine on the carboxyl that exposes on the polymer.It is that pH is 7.2 phosphate buffered saline (PBS) (PBS) or 0.5 mole 2-(N-morpholino) ethyl sulfonic acid that two (amine) Polyethylene Glycol (PEG) provides amine groups, buffer.This solution and electrostatic spinning pipe be incubation one hour at room temperature.
10. with the described sample of distilled water cleaning down.
11. use EDC, Sulfo-NHS and buffer finally are connected to the amine groups on two (amine) PEG on the carboxyl of hirudin.This solution and electrostatic spinning pipe be incubation two hours at room temperature.
12. sample PBS cleaning down.
13. dry sample or air-dry under vacuum, and be kept under the aseptic condition.
14. before operation is implanted, graft was placed in the Sterile Saline 1-minute.
The zooscopy that relates to biomimetic scaffolds
[0309] all methods are all by in the University of California, and the institutional evaluation committee in Los Angeles and the care of animal and use committee get permission.Athymism rat (6-8 week age, about 180g) is available from the animal department of National Cancer Institute.Rat is with 2.0% isoflurane anesthesia and the placement of lying on the back.Make little otch to expose and separation left common carotid artery (CCA).Graft end-right-end is placed in clamp and ligation, and with 10-0 line interrupted suture.Before implanting or during do not use heparin or any other anticoagulant at any point.The taking-up of graft relates to the initial step identical with being used for implantation process.The heparin of 40 units is expelled to external jugular vein, to prevent during taking out implant, blood coagulation taking place.By graft being taken out closing on the stitching thread position natural CCA of direct ligation.In order to carry out histologic analysis, sample put into OCT and at-20 ℃ of freezings.Make the frozen section of 10 μ m thickness.Use following primary antibody to carry out immunohistochemical staining and analyze section.CD31 (BD Biosciences), myoglobulin heavy chain (SantaCruz Biotech) and CD68 (Chemicon).(Dako, Carpinteria CA) replace primary antibody to be used for negative control to use mice IgG isotype.With Zeiss Axioskop 2MOT microscope photographing immunohistochemistry image.
[0310] makes the common carotid artery 30 days do not comprise the PLLA graft of covalently bound biomolecule (being hirudin) and to be implanted to Sprague Dawley rat (about 180grams).Before implantation, during or do not use any systemic anti-platelet agents, anticoagulant or heparin afterwards in any site.The dyeing of the Verhoeff ' s elasticity of Figure 42 A shows that graft has that tangible inside is reinvented, thrombosis and neointimal hyperplasia.By comparing with the PLLA graft, thrombosis (dyeing is darkviolet) obviously, internal film tissue's (leave graft and towards the intracavity growth).Figure 42 B has shown the monolayer endothelial cell that adheres to the thrombosis tissue and leave the fusion of PLLA graft.The smooth muscle cell specific stain (myoglobulin heavy chain) of Figure 42 C shows neointimal hyperplasia and definite sign of inwardly reinventing.Figure 42 D demonstration is compared with the hirudin graft, and the quantity of mononuclear cell and macrophage (CD68) increases slightly and is present on the wall of graft.
[0311] graft puted together of two hirudins of preparation and the common carotid artery that is implanted to Sprague Dawley rat (about 180g) are 28 days.Before implantation, during or do not use any systemic anti-platelet agents, anticoagulant or heparin afterwards in any site.The brazilwood extract dyeing of Figure 43 A shows that 100% is unimpeded, and intracavity does not have thrombosis.Figure 43 B shows the monolayer endothelial cell (CD31) of the fusion that directly adheres to PLLA nanofiber net surface, and does not obviously have thrombosis on the graft.The smooth muscle cell specific stain (myoglobulin heavy chain) of Figure 43 B shows does not have the neointimal hyperplasia phenomenon.Among Figure 43 D the quantity of mononuclear cell and macrophage (CD68) seldom, and only limit to the PLLA graft around.
[0312] Verhoeff ' s elasticity staining kit is bought from AmericanMaster*Tech Scientific Inc., is used to show elastic fiber and collagen fiber.It is 7 μ m that the freezing sample of OCT embedding is frozen section.The pictorial display elastic fiber is a black, and collagen fiber are red.Verhoeff ' s working stain is the mixture of hematoxylin, ferric chloride and iodine solution, and every kind of sample was immersed wherein 15 minutes.Use 2% ferric chloride differentiation solution then 3 minutes, and then sample was put into Van Gieson ' s dyeing 30 seconds.Microscope slide is through a series of gradient alcohol dehydration, transparent and with the sealing of capping glue with Safeclear II.
H and E dyeing: Harris method
Material: all be stored in the red Flammable case.
| Title | Catalogue # |
| Histoprep ethanol (ethanol) | Fisher?HC800 |
| Safeclear II (dimethylbenzene displacement) | EXPERIMENTAL DESIGN 044-192 |
| Eosin Y | EXPERIMENTAL DESIGN 316-631 |
| The Harris hematoxylin | EXPERIMENTAL DESIGN 245-678 |
| Dyeing Bluing Solution | Harleco?65354185 |
Experimental design
In distilled water, wash microscope slide.
Immersed the Harris hematoxylin 3 minutes.
Wash up to transparent with tap water, totally 3 times, 10 seconds.
In Bluing Solution, dyed blue 1 minute.
Wash up to transparent 10 seconds with tap water.
Redyed 30 seconds with Yihong.
Use the gradient ethanol dehydration: 95%, 100%, 100%, each is 1 minute.
With dimethylbenzene (or Safeclear II) dehydration, each is 5 minutes.
With resin encapsulation medium (Permount) sealing.
Dispose chemical waste product: H, E, alcohol.
Observe: (H: purple=nuclear; E: pink colour=substrate, cytosol)
[0313] is appreciated that embodiment described herein and embodiment only are for exemplary purpose, those skilled in the art will obtain the various modifications under its instruction or the enlightenment of variation, and be included in the scope of the application's spirit and limit and claims.All publications that this paper quoted, patent and patent application are incorporated this paper into by reference for all purposes.
Claims (40)
1. compositions comprises: the pipe of the first cellulosic polymers support tube or filling, and hirudin, one or more fiber of the wherein said first cellulosic polymers support is aligned; Wherein said hirudin is directly or by the non-covalent connection of junctional complex or covalently bound to the described first cellulosic polymers support.
2. according to the compositions of claim 1, the wherein said first cellulosic polymers support has and is selected from following length: about 0.01cm is to about 20cm, about 0.05cm is to about 5cm, about 0.5cm is to about 5cm, about 1cm is to about 5cm, and about 2cm is to about 5cm, and about 1cm is to about 3cm, about 2cm arrives about 15cm to about 10cm and about 5cm.
3. according to the compositions of claim 1, the wherein said first cellulosic polymers support is substantially along being selected from vertically and the arrangement of the direction of circumference.
4. according to the compositions of claim 1, the wherein said first cellulosic polymers support has seam.
5. according to the compositions of claim 1, the wherein said first cellulosic polymers support is seamless.
6. according to the compositions of claim 1, the wherein said first cellulosic polymers support is integrally formed.
7. according to the compositions of claim 1, at least one fiber of the wherein said first cellulosic polymers support comprises and is selected from following polymer or subunit: aliphatic polyester, polyalkylene oxide, polydimethylsiloxane, polycaprolactone, polylysine, collagen, laminin, fibronectin, elastin laminin, alginate, fibrin, hyaluronic acid, Dan Baijutang, polypeptide and combination thereof.
8. according to the compositions of claim 7, wherein said aliphatic polyester is selected from as follows: lactic acid (D-or L-), lactide, poly-(lactic acid), poly-(lactide), hydroxyacetic acid, poly-(hydroxyacetic acid), poly-(Acetic acid, hydroxy-, bimol. cyclic ester), Acetic acid, hydroxy-, bimol. cyclic ester, poly-(lactide-copolymerization-Acetic acid, hydroxy-, bimol. cyclic ester), poly-(lactic acid-copolymerization-hydroxyacetic acid), and combination.
9. according to the compositions of claim 7, wherein said polyalkylene oxide is selected from as follows: poly(ethylene oxide), poly(propylene oxide), and combination.
10. the compositions of claim 1, at least one fiber of the wherein said first cellulosic polymers support comprise that poly-(lactide-co-glycolide) (PLGA) or poly-L-lactide (PLLA).
11. the compositions of claim 1, wherein said hirudin is covalently bound to the described first cellulosic polymers support by junctional complex.
12. comprising, the compositions of claim 1, wherein said junctional complex be selected from following member: peptide, saccharide, poly-(ether), poly-(amine), poly-(carboxylic acid), poly-(aklylene glycol), poly-(ethylene glycol), poly-(propylene glycol), the copolymer of ethylene glycol and propylene glycol, poly-(oxyethylatedpolyol), poly-(enol), poly-(vinylpyrrolidone), poly-(hydroxypropyl methyl acrylamide), poly-(alpha-hydroxy acid), poly-(vinyl alcohol), poly-phosphorus piperazine, poly-oxazoline, poly-(N-acryloyl morpholine), Polysialic acid, polyglutamic acid, poly-aspartate, polylysine, polymine, polyactide, polyglycerin ester and copolymer thereof and polyacrylic acid.
13. comprising, the compositions of claim 11, wherein said junctional complex be selected from following member: poly-(ethylene glycol), poly-(propylene glycol) and combination thereof.
14. the compositions of claim 1 further comprises around the first cellulosic polymers support tube or fills the outer surface of pipe but be not positioned at the first cellulosic polymers support tube or fill the sleeve of the intracavity of pipe.
15. comprising, the compositions of claim 14, wherein said sleeve be selected from following polymer or its subunit: according to ethylene terephthalate and politef.
16. the compositions of claim 14, wherein said sleeve comprise the second cellulosic polymers support, and described second cellulosic polymers support alignment or have random orientation.
17. the compositions of claim 16 also comprises around first sleeve of first end of the first cellulosic polymers support with around second sleeve of second end of the first cellulosic polymers support.
18., also comprise cell according to the compositions of claim 1.
19. according to the compositions of claim 18, wherein said cell embedding is in the first cellulosic polymers support or on the surface of the first cellulosic polymers support.
20. according to the compositions of claim 18, wherein said cell is selected from stem cell and CFU-GM.
21. according to the compositions of claim 18, wherein said cell is selected from as follows: the vascular cell of growing up, blood vessel CFU-GM, blood vessel stem cell.
22. according to the compositions of claim 1, said composition is applied on the rotation mandrel by the polymer solution that will contain polymer and makes.
23. according to the compositions of claim 1, wherein said polymer support makes by the electrostatic spinning process that comprises the rotation mandrel with at least one non-conductive area.
24. according to the compositions of claim 1, the wherein said first cellulosic polymers support is seamless, the periphery alignment, and at least one fiber of the described first cellulosic polymers support comprises poly-(lactide-co-glycolide) (PLGA); Described hirudin is covalently bound to the described first cellulosic polymers support by junctional complex, and described junctional complex is to be selected from following member: diaminourea poly-(ethylene glycol) and poly-(ethylene glycol).
25. the compositions of claim 24, wherein said compositions have the length of about 1mm to about 50cm.
26. the compositions of claim 24, wherein said compositions have the length of about 0.5cm to about 10cm.
27. the compositions of claim 24, wherein said compositions have the length of about 3mm to about 6mm.
28. the compositions of claim 24, wherein said compositions have the internal diameter of about 0.01mm to about 6mm.
29. the compositions of claim 24, wherein said compositions have the internal diameter of about 3mm to about 6mm.
30. the compositions of claim 29, wherein said compositions have the length of about 4cm to about 8cm.
31. the compositions of claim 24, wherein said compositions is used as: A/V diverter or hemodialysis access graft.
32. pharmaceutical composition, it comprises:
(a) compositions of claim 1; With
(b) pharmaceutically acceptable excipient.
33. the method for treatment experimenter damage, described method comprises:
(i) with the compositions of claim 1 or 24 with the amount that is enough to treat described damage and be enough to treat the target site that is administered to described experimenter under the condition of described damage.
34. according to the method for claim 33, wherein said target site is selected from as follows: coronary artery, femoral artery ， popliteal tremulous pulse, carotid artery, cerebral arteries, abdominal part, above knee, the following and radius of knee.
35. according to the method for claim 33, wherein said target site is selected from carotid artery and the following vascular tissue of knee.
36. method according to claim 33, wherein said damage relates to the blood vessel of disconnection, the described first cellulosic polymers support has the pipe that comprises first end and second end or fills the shape of pipe, and the blood vessel of described disconnection comprises the first blood vessel stump and the second blood vessel stump, and described using comprises:
(ii) described first end with described compositions is connected to the described first blood vessel stump; With
(iii) described second end with described compositions is connected to the described second blood vessel stump.
37. strengthen the method for experimenter's angiogenic growth, described method comprises:
(i) with the compositions of claim 1 or 24 with the amount that is enough to strengthen angiogenic growth and be enough to strengthen the target vascular site that is administered to described experimenter under the condition of angiogenic growth.
38. the method for compositions of preparation claim 1.
39. the method for compositions of preparation claim 1, described method comprises:
(i) one or more fiber is carried out the electrostatic spinning process, thereby make described compositions.
40. according to the method for claim 39, wherein said electrostatic spinning process comprises the rotation mandrel with at least one non-conductive area.
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US80435006P | 2006-06-09 | 2006-06-09 | |
| US60/804,350 | 2006-06-09 | ||
| US60/861,780 | 2006-11-30 | ||
| USPCT/US2007/016253 | 2007-01-29 | ||
| US11/668,448 | 2007-01-29 | ||
| US60/924,926 | 2007-06-05 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101500508A true CN101500508A (en) | 2009-08-05 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007800292629A Pending CN101500508A (en) | 2006-06-09 | 2007-06-11 | Biomolecule-linked biomimetic scaffolds |
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| Country | Link |
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| CN (1) | CN101500508A (en) |
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