CN101668552A - Implantable material for medical or surgical application - Google Patents

Abstract

Implantable materials for medical or surgical applications comprising specific chemical groups on their surface to alter the physico-chemical properties of said material rendering it suitable implantation or biocompatible properties.

Description

The implantable material that comprises cellulose and glycopeptide xyloglucan-GRGDS

Technical field

The present invention relates to medical treatment or the operation usefulness implantable material, its comprise change described material physicochemical properties to give the particular chemical group of its suitable implantation character or biocompatibility.More specifically, the present invention relates to comprise polymer carbohydrate implantable material, prepare the method for these implantable materials and the purposes of the material made by these methods and comprise the product of these materials.

Background technology

Organ or tissue's depletion is serious health problem.Organizational project provides to be used from the functional health cell (that is, from body, allos or heterogenous cell) of separate sources and/or the extracellular is natural or synthetic polymer is rebuild the potentiality of function of organization.

Have various a large amount of synthesizing polymeric materials of different nature and nowadays in medical treatment and beautifying use, use, as prosthese, implant and the support of used in tissue engineering.These synthesizing polymeric materials can be divided into two primary categories usually: temporary implant/can biology absorbs again and long-term implant/can not absorb again by biology.Example that can biological resorbent synthetic material comprises the polymer that comprises poly-l-lactic acid (PLLA) and poly-l-glycolic (PLGA).Long-term implantable and can not the biological example of absorbing material again be poly-(tetrafluoroethene) PTFE, it be used in the implantable goods of multiple medical treatment, comprises in blood vessel graft and tissue repair sheet material and the sticking patch.

But these synthetic materials also have restriction and shortcoming, cause foreign body reaction in vivo as the improper interaction between polymer and the cell, as inflammation, infection, aseptic loosening, local organization refuse and implant encapsulation and thrombosis and thromboembolism.Therefore, the success of promising polymer depends in part on relevant cell adhering to and grow in its surface.The surface chemistry mediated cell is replied and is influenced cell in this lip-deep adhesion, propagation, migration and function this material.

In arteries reconstruction field, more and more need functional minor diameter artificial graft thing (internal diameter＜6 millimeter).When self replaces the blood vessel non-availability (for example because the vascular system situation of patient's difference), the surgeon has no option except that implanting synthetic polymer base blood vessel.After implantation, be by almost blocking immediately that blood coagulation and platelet deposition cause under low relatively flox condition about the initial subject matter of these blood vessels.Owing to cause the thrombotic interaction of bringing out property of surface to occur in blood-biomaterial at the interface, surface modification has been to improve the mode of blood compatibility.In different modifications, also be widely used in the thrombosis that makes on the artificial organ with the modification of polysaccharide (as heparin) and minimized.But problem is that when heparin directly was attached to surperficial going up, the active of heparin significantly reduced.So far, the biocompatible surface of successful type is that end points connects (end-point-attached) heparin surface.

Another attractive surface modification is to introduce to prevent plasma proteins absorption, platelet adhesion and thrombotic hydrophilic radical.Problem is to make them to be retained on the surface of long-term device (as blood vessel graft).Especially with water-soluble polymer, as polyacrylamide (PAAm); Gather (N,N-DMAA) (PDMAAm); Poly-(ethylene glycol) (PEG), ethylene-vinyl alcohol copolymer (EVA) and gather (methacrylic acid 2-hydroxyl ethyl ester) and (PHEMA) be transplanted on the surface of solids to prevent protein adsorption.

Material modification must be managed the mimic biology inactive surfaces with other theory of improving its blood compatibility.In blood vessel, this inactive surfaces is made of monolayer endothelial cell.At present as the material of blood vessel graft, can not promote the adhesion or the propagation of human endothelial cells as expanded PTFE (ePTFE) and polyester.Therefore carry out surface modification with fibrinogen, Fibronectin or with fixed RGD (Arg-Gly-Asp) (it is an attachment proteins, as the minimal segment of the avtive spot of fibrinogen, Fibronectin and the von Willbrand factor).Except that with the heparin surface modification, big quantity research and test have been carried out so that endotheliocyte is inoculated on the PTFE tube.But problem is to make cell long-period to keep from the teeth outwards.

But, these synthetic materials mentioned above, for example PTFE and polyester also have other restriction and shortcoming, comprise narrow physics and biochemical property.Therefore, still need to seek the substituting implantable material that more is applicable to particular surgical application.

Some investigators have studied the tissue biocompatibility of cellulose and derivant thereof and have examined some application-specific of this material.Particularly, investigated and to be used in the organizational project by the cellulose of microorganisms.The microbial source cellulose has network structure, wherein tangled each other by the very thin ribbon-like fibre intricately that constitutes of cellulose of highly crystalline and height uniaxial orientation, and this network structure contains big quantity of fluid in the portion space within it.Because this cellulose is made of the many ribbon-like fibres with high-crystallinity, even this cellulose also can be resisted external force such as tensile force under wet condition.Micro organism cellulose is not structurally to be different from plant-derived cellulose, but does not find the structure such as the above-mentioned structure of high-sequential in the plant source cellulose, and it is the characteristic of micro organism cellulose.Correspondingly, although micro organism cellulose is gel, has high strength.

US 6800753 has described use regenerated cellulose (RC) and oxidized regenerated cellulose (ORC) preparation used in tissue engineering support.RC and ORC composite by at first with cellulose dissolution in dicyandiamide solution, the required supporting structure of this cellulose regenerated one-tenth is prepared.In order to make porous support, in this dicyandiamide solution, introduce pore former in supporting structure, to produce the hole.Can be subsequently with this support oxidation to introduce carboxyl, aldehyde and/or ketone in its surface.These functional groups serve as cell adhesion or further the site of chemical modification adhere to inducing cell and propagation subsequently.

People (" Alginate microcapsules prepared with xyloglucanas a synthetic extracellular matrix for hepatocyte attachment " such as Seo S, Biomaterials, the 26th volume no.17 (2005), 3607-3615 page or leaf) described with the calcium alginate polymer carbohydrate capsule of xyloglucan (XG) modification to prepare the synthetic cell epimatrix of former generation mouse liver cell.The liver specificity function that improves interacts owing to galactose moiety and the specificity between the asialoglycoprotein receptor on the hepatocyte of XG.

People (" Biodegradable scaffolds-delivery systems forcell therapies " such as Yang Y, Expert Opinion on Biological Therapy the 6th volume, no.5 (2006), the 485-498 page or leaf) be to discuss to use the survey article of biomolecule with the Biodegradable material surface modification.It is important that biodegradable support it is said in the application facet that makes things convenient for cell therapy.Discussed the selection of timbering material, particularly aspect biocompatibility.Summarized the manufacturing of the formation of biomimetic scaffolds, the new manufacture that can control Framework construction and micro structure and injectable and in-situ cross-linked support.Summarized providing and still had challenge aspect the Biodegradable stents that can guide the cell that it contacted rightly.

People (" Xyloglucan and xyloglucan endotransglycosy-lases (XET): Tools for ex vivo cellulose surface modification " such as Zhou Q, Biocatalysis and Biotransformation, the 24th volume, no.1-2 (2006), the 107-120 page or leaf) be survey article in the wood fiber technical field, the modification of having described the wood fiber is to provide novel biomaterial and cellulosic novel surface method of modifying.Provide to be used to make functional group to be connected to new system on the wood pulp, comprise with the high-affinity between xyloglucan and the cellulose interact, glycosyl transferase catalysis polysaccharide-peculiar property of oligosaccharide coupling reaction and the synthetic combination of traditional carbohydrate in the xyloglucan.

But, having good biocompatibility providing, still there is challenge in the implantable material aspect that especially has the surface that comprises bioactie agent.

There are variety of issue in the regenerated cellulose of making according to US 6800753 (RC) and the use of oxidized regenerated cellulose (ORC).For example, may be unsatisfactory with the solvent processing cellulose, because this processing can be complicated because of the structural change of cellulosic material.These structural change meetings cause shrinking and the form that changes and obtain more crisp material.

At present, as mentioned above, implantable material is limited by physics and the relevant problem of biochemical property (as their surface chemistry) with them.The previous long-term existence of order but still unsatisfied needs are the new implantable materials of exploitation and improve their character undoubtedly, so that they can successfully be used in the medical applications.

Correspondingly, main purpose of the present invention provides to be used to prepare to be had and the improving one's methods of the implantable material of the fabulous compatibility of live body, and the implantable material made from this method is provided.

Another object of the present invention provides and has the expectation engineering properties the implantable material of (as machinery and hot strength, elongation and the property sewed up).

Another object of the present invention provides the implantable material of other factor that has the cell attachment site or influence cell adhesion, propagation, migration and function.

A further object of the present invention provides the implantable material that is applicable to that body is implanted into.

From describing, following invention can clear find out the ways and means of realizing each above-mentioned purpose and other purpose.

Summary of the invention

For realizing purpose of the present invention, the inventor provides the new method for preparing implantable material, comprise that wherein said chemical group is given this PCM improved biocompatibility by the carbohydrate link molecule (CLM) that comprises chemical group is bonded on the polymer carbohydrate material (PCM) the PCM modification.

It is to avoid chemical treatment PCM that preparation of the present invention comprises with one of major advantage of the method for the implantable material of the PCM of chemical group modification.This processing can change conformation (confirmation) or orientation and other physicochemical properties of PCM.Therefore, method of the present invention has been avoided the fibre structure that often runs into and the loss of performance in the direct chemical modification of PCM.

For example, because with an organic solvent, the chemical modification of hydrogel such as Bacterial cellulose can be complicated because of structural change.By aqueous chemical enzymatic technology being used for the surface modification of Bacterial cellulose, the present invention has avoided these structural changes.

In others of the present invention, the implantable material that provides the method according to this invention to make; This implantable material is used to make the purposes of used in tissue engineering support; The used in tissue engineering support that comprises material manufactured according to the present invention; With in-vivo tissue displacement and/or renovation process.

According to a specific embodiments, the present invention includes the artificial blood vessel that comprises implantable material manufactured according to the present invention.Artificial blood vessel of the present invention is characterised in that high penetration resistance, high burst pressure and excellent biological compatibility.

Especially with reference to accompanying drawing the present invention is described in more detail below.

Description of drawings

Fig. 1 shows unmodified polymer carbohydrate material (PCM) (1) and modification PCM (6).Modification PCM comprises carbohydrate link molecule (CLM) (2), and described CLM (2) comprises at least a portion solubility polymerization carbohydrate (SCP) (3) and chemical group (5) and the optional compound carbohydrate polymer fragment (CPF) (4) that comprises this chemical group.

Fig. 2 illustrates and uses enzyme and CPF (4) preparation CLM (2).SCP (8) is contacted with the CPF that comprises chemical group (5) (4) with enzyme (7).This enzyme (7) makes the SCP fracture and introduces the CPF that this contains chemical group, obtains product C LM (2).

Fig. 3 illustrates with Direct Red 28 (Congo red) painted Bacterial cellulose ▲ and velveteen ● Langmuir adsorption isotherm (A).Line among the figure B is represented results of linear regression analysis.

Fig. 4 illustrates and is adsorbed with xyloglucan ● and xyloglucan-GRGDS ▲ the Langmuir adsorption isotherm (A) of Bacterial cellulose.Line among the figure B is represented results of linear regression analysis.

Fig. 5 illustrates and is adsorbed with xyloglucan ● and xyloglucan-GRGDS ▲ the Langmuir adsorption isotherm (A) of velveteen.Line among the figure B is represented results of linear regression analysis.

Fig. 6 illustrates xyloglucan ● and xyloglucan-GRGDS ▲ adsorbance and the functional relationship of the specific surface area of cellulosic substrate.This line is represented results of linear regression analysis.

Fig. 7 illustrates the degree of crystallinity of Bacterial cellulose, velveteen and disappearing fibre (lyocell).

Fig. 8 illustrates the chemical constitution of GRGDS xyloglucan oligosaccharide (XGO-GRGDS).

Fig. 9 show cell culture medium be adsorbed onto (-) on the cellulose, xyloglucan then cell culture medium be adsorbed onto (...) on the cellulose, xyloglucan-GRGDS then cell culture medium be adsorbed onto the QCM adsorption isotherm of (-) on the cellulose.Arrow representative washing.

Figure 10 shows unmodified Bacterial cellulose (A), the optical microscopic image of the ECs of the Bacterial cellulose (C) of Bacterial cellulose of xyloglucan modification (B) and xyloglucan-GRGDS modification.

Figure 11 shows untreated Bacterial cellulose (A), the Bacterial cellulose (B) after handling in acetone and with the SEM image of the Bacterial cellulose (C) of xyloglucan-GRGDS modification.

The specific embodiment

The present invention relates to develop the implantable polymer carbohydrate material (PCM) of medical treatment or operation usefulness, comprise the particular chemical group in its surface to change the physicochemical properties of described material.Especially, described chemical group is given this PCM improved biocompatibility, for example the attachment site by cell is provided or influence cell other factor in this lip-deep adhesion, propagation, migration and function.

In addition, the present invention relates to prepare the method for implantable material of the present invention and the purposes of the material made by these methods and comprise the product of these materials.

In this manual, unless indicate separately, " one " or " a kind of " is meant " one or more ".

About the present invention, term " biocompatibility " relates to the character of material, i.e. the compatible character of material and live body.In other words, coordinate with life entity; Biological function there is not influence poisonous or infringement.If for example bring out untoward reaction when material is contacted with the part of live body, then this material and live body have the poor compatibility.This untoward reaction meeting causes foreign body reaction, as inflammation, infection, aseptic loosening, local organization refuse and implant encapsulation and thrombosis and thromboembolism.On the contrary, if this badness reaction does not take place, then observe excellent compatibility with live body.In addition, improved biocompatibility means the improved compatibility of material and live body.The example of biocompatibility is a blood compatibility, i.e. the character of material and blood compatibility.

The method for preparing implantable material

According to a first aspect of the invention, the method that polymer carbohydrate material (PCM) modification is prepared implantable material by making the carbohydrate link molecule (CLM) that comprises chemical group be bonded to that PCM goes up is provided, and wherein said chemical group is given this PCM improved biocompatibility.

An embodiment of this method is shown among Fig. 1, it shows unmodified PCM (1) and carbohydrate link molecule (CLM) (2), and described CLM (2) comprises at least a portion SCP (3) and chemical group (5) and the optional compound carbohydrate polymer fragment (CPF) (4) that comprises this chemical group.Because this CLM can be bonded on the PCM, so bonding takes place when PCM is contacted with CLM.

In one embodiment of the invention, this method comprises the following steps: that (a) provides the carbohydrate polymer fragment that comprises chemical group (CPF); (b) the described CPF that comprises this chemical group and solubility polymerization carbohydrate (SCP) are contacted under the condition that causes forming the complex of being made up of described CPF that comprises this chemical group and SCP, described CPF and SCP form carbohydrate link molecule (CLM) together; (c) described complex and the PCM that wants modification in that being bonded under the condition on the PCM, this complex are contacted.

In a preferred embodiment, CLM contact PCM that comprises chemical group and the step that is bonded on it are carried out under aqueous conditions.Such advantage is, makes the CLM contact and is being bonded to that the PCM form does not change in the step on the PCM.

Preparation comprises the method for CLM of chemical group, promptly prepares the method for the complex of being made up of described CLM and described chemical group and PCM branch and opens and carry out.Thus, the preparation method of CLM that comprises chemical group can enlarge scale and comprise several steps and harsh condition.

The term " polymer carbohydrate material " of being abbreviated as " PCM " relates to the material that comprises water-fast polymer carbohydrate material and/or water-soluble polymeric carbohydrate materials, and its all or part of repetitive by one or more monosaccharide is formed.This class PCM normally contains two or more dissimilar polymer carbohydrates or carbohydrate containing polymer and another polymer such as proteinic composite.

According to the present invention, PCM is suitable as implantable material, for example is used as any polymer carbohydrate material of the key component of used in tissue engineering support.Available different PCM for example are described among the WO 03/033813 among the present invention.

In a specific embodiments, this PCM is the cellulosic material form, promptly comprises cellulose.In the present invention, cellulose can extract from annual plant (as Caulis et Folium Lini, Fructus Cannabis or corn) or perennial plant (as cotton, Cortex Populi dividianae, birch, willow, Eucalyptus, Larch, pinaster or PiceameyeriRehd. Et Wils.).The example of suitable cellulosic material comprises purification cotton, velveteen, alpha-cellulose, wood pulp, pure wood pulp, powdered cellulose, microcrystalline Cellulose and/or is modified as the cellulose of other polymer.

Compare with plant cellulose that to have another cellulose source of different nature be the microbial source cellulose.The microbial source cellulose is being as receiving publicity in the biomaterial, this mainly due at given application with its be molded as difform probability with and biocompatibility and high-purity.This cellulose is extracellular polysaccharide and the manufacturing quite cheaply by cultivating acetobacter xylinum (Acetobacterxylinum).Compare with plant cellulose, this cellulose with its pure form extrude and not with any other polymer or protein bound.Bacterial cellulose can be with the effective purification of sodium hydroxide, to realize the endotoxin value of FDA about the graft that contacts with blood, promptly every device＜20EU.Bacterial cellulose contains 99% water and is regarded as (although not being by definition) hydrogel.Although its solid content is low, the fibriilar component network of nano-cellulose provides good mechanical performance (mechanics).

Term " microbial source cellulose " relates to the cellulose that is produced by microorganism (as antibacterial) as mentioned above.In a preferred embodiment of the invention, use the culture of the synthetic antibacterial of cellulose (belonging to) as acetobacter xylinum.

The term " soluble-carbohydrate polymer " of being abbreviated as (SCP) relates to the polymer that comprises one or more different monosaccharide or derivatives thereofs, and it may be dissolved in aqueous or the organic solvent.Example comprises the polysaccharide that classifies as hemicellulose (just by those carbohydrate polymers that β (1-4)-the connection glucose unit is formed, i.e. cellulose), pectin (polyuronic acid and ester) and starch (contain or do not contain the ramose α of α (1-6) side chain (1-4)-be connected polydextrose).Xyloglucan is by the polysaccharide that the β (1-4) that has α (1-6) xylose residues-connection polydextrose skeleton is formed, and wherein α (1-6) xylose residues itself can further be replaced as fucose and arabinose by other sugar; It is the example of this class SCP, particularly is hemicellulose.In a preferred embodiment, SCP can be bonded on the PCM, for example via one or more hydrogen bonds, ionic interaction, one or more covalent bond, Van der Waals for or these any combination.In one embodiment of the invention, SCP can be according to CPF described below.In a preferred embodiment, SCP comes from xyloglucan (XG).

" come from xyloglucan " and be meant by having α (1-6) the xylose residues β (1-4) of (this α (1-6) xylose residues itself can be further replaced by other sugar as fucose and arabinose)-is connected the polysaccharide that the polydextrose skeleton is formed, and the version and the fragment of further chemical replacement and modification.

It can be enzymatic preparation or the segmental molecule of chemical preparation of SCP that the term " carbohydrate polymer fragment " of being abbreviated as " CPF " relates to.The segmental example of this class comprises the repetitive of any amount of described SCP.

Therefore suitable fragment can contain 2 to about 5000 monosaccharide units, as 2-10,4-10,3-100,11-15,20-25,26-40,41-60,61-100,101-200,201-300,301-400,401-500,501-1000,1001-2000,2001-3000, a 3001-4000 or 4001-5000 monosaccharide unit in polymer backbone.This CPF can further comprise the side chain with different length and composition.Instantiation includes but not limited to xyloglucan (xylogluco)-oligosaccharide (XGO) or its fragment, or with one or more fucosyl residues or the further modification of other monosaccharide.

XGO is generally according to people such as Fry (1993) Physiologia Plantarum, 89, the nomenclature name of summarizing among the 1-3, wherein G represents unsubstituted β-glucopyranosyl residue, X represents xylopyranosyl-α (1-6) glucopyranosyl unit, L represents galactopyranose base-β (1-2)-xylopyranosyl-α (1-6) glucityl unit, and F represents pyrans fucosido-α (1-2)-galactopyranose base-β (1-2)-xylopyranosyl-α (1-6)-glucityl unit.These different units can connect between the glucopyranosyl unit to form β (1-4)-glucosan polysaccharide skeleton via β (1-4) connecting key.Using this nomenclature, is XXXG, XLXG, 25XXLG and XLLG the tamarind xyloglucan being carried out the endoglucanase digestion isolated XGO in back usually.If the reducing end glucose (G) of these oligosaccharide is reductive sugar alcohol forms, this unit is represented with " Gol ".Therefore, for example be designated as XXXGol, XLXGol, XXLGol and XLLGol from reduction (sugar alcohol) derivant of the above-mentioned oligosaccharide of tamarind xyloglucan.

Preferably, CPF is derived from xyloglucan and can contain 3 to 100, comprises 4 to 10 polymer backbone monosaccharide units.

The term " carbohydrate link molecule " of being abbreviated as " CLM " relates to molecule or the complex to small part and chemical group that contains with good grounds above-described SCP.CLM can be bonded on the PCM, for example via one or more hydrogen bonds, ionic interaction, one or more covalent bond, Van der Waals for or these any combination.

CLM can be by organic or chemosynthesis and/or by utilizing the catalytic activity preparation of some enzyme.

In one embodiment of the invention, CLM can utilize the method preparation described in the WO 2004/094646Al.For example, this CLM that comprises chemical group can prepare by the method that comprises the following steps: by xyloglucan polymer manufacture xyloglucan fragment; With one or more chemical groups are connected on segmental reducing end of this xyloglucan and/or the side chain, make the CLM that comprises chemical group that can be used for being bonded on the PCM thus.

The embodiment of using enzyme and CPF to prepare CLM is shown among Fig. 2.SCP (8) is contacted with the CPF that comprises chemical group (5) (4) with enzyme (7).In this embodiment, enzyme (7) makes the SCP fracture and introduces the CPF that this contains chemical group, thereby produces product C LM (2).

This CLM can comprise one or more chemical groups.

In one embodiment of the invention, this CLM can use and the enzyme that the monosaccharide or the oligosaccharide of natural or chemical modification are transferred on oligosaccharide or the polysaccharide end can be prepared.This fermentoid includes but not limited to have high glycosyl transfer activity but the enzyme of low hydrolysing activity, have the glucityl hydrolytic enzyme of high intrinsic glycosyl transfer activity, through engineering approaches is with the enzyme of the glycosyl transfer activity that improves them with use the glucosyltransferase of nucleotide sugar as substrate.

In WO 03033813 more detailed description according to the present invention spendable different enzyme and character thereof and how to obtain described enzyme.

In a preferred embodiment of the invention, this enzyme is a glycosyl transferase (XET, EC 2.4.1.207) in the xyloglucan.

In nature, glycosyl transferase such as XET enzyme play a role in the living plant body, so this enzyme obviously can be worked in aqueous environments.Therefore method of the present invention can be implemented in aqueous solution, or it can be implemented in the presence of some component such as buffer agent and/or wetting agent and/or stabilizing agent and/or polymer and/or the reduction active organic component of water (as DMSO) in water.About the further details of these components, referring to WO 03/033813.

According to specific embodiments of the present invention, can the XGO of chemical modification be coupled on the xyloglucan (XG) by at first in solution, using the XET enzyme, modification XG is adsorbed onto on the cellulosic material, new chemical group is added in the cellulosic material that does not contain intrinsic xyloglucan.

In the present invention, term " chemical group " relates to any chemical group of the potential PCM of can be used for modification.Modification is meant the functional character that changes PCM.The ability that changes the functional character of PCM is inherent in this chemical group on this meaning.According to the present invention, this PCM is given in this modification improved biocompatibility.Therefore, this chemical group changes the physicochemical properties of described PCM so that its biocompatibility is better.

Biocompatibility relates to the material character compatible with live body.A kind of mode of improving the biocompatibility of material is that influence contacts with this material or adhesion, growth, migration, propagation, differentiation, shape, polarity and/or the metabolic function of interactional cell.A kind of concrete mode of improving the biocompatibility of material is to reduce the trend that this material brings out the blood coagulation that contacts with this material.For material used in the artificial blood vessel, this anticoagulation character is particularly useful.

Contained change PCM physicochemical properties in the chemical group of the present invention so that its improved biocompatible composition include but not limited to: anticoagulin, ECM adhesion molecule, somatomedin, cell adhesion molecule and adhesin polypeptide fragment and cell culture substrate and cell nutritious element.It will be apparent to those skilled in the art that this composition is enumerated is not limit, can use the physicochemical properties that change PCM so that other suitable component of its improved biocompatible in the present invention.

In the present invention, term " anticoagulin " relates to the molecule that reduces blood coagulation trend.The preferred embodiment that is used for this quasi-molecule of the present invention is the pentasaccharides inhibitor of heparin, low molecular weight heparin and Xa factor, reaches heparin and Ai Zhuo heparin (idraparinux) as sulphur.

About the present invention, term " ECM adhesion molecule " relates to the extracellular macromole that constitutes extracellular matrix (ECM).These macromole (mainly being protein and polysaccharide) merocrine secretion goes out and be assembled into organized 3-D network in the extracellular space of majority tissue.The ECM molecule comprises glycosaminoglycans and Dan Baijutang, as chondroitin sulfate, Fibronectin, heparin sulfate, hyaluronidase (hyaluron), dermatan sulfate, keratin sulfate, laminin, collagen protein, heparan sulfate proteoglycan and elastin laminin.Extracellular matrix is regulated tissue, cell differentiation and the cell of cell within a cell skeleton and the spatial configuration of tissue.In fact, ECM regulates at the cell development that is in contact with it by influence, migration, propagation, differentiation, shape, polarity and metabolic function and plays a key effect aspect the behavior of this cell.

About the present invention, term " somatomedin " relates to the biologically active polypeptide that causes cell proliferation.They include but not limited to that epidermal growth factor, transforming growth factor, nerve growth factor, acidity and basic fibroblast growth factor and angiogenesis factor, platelet derived growth factor, insulin and insulin like growth factor comprise somatomedin, myxoma and vaccinia virus source somatomedin.

About the present invention, term " cell adhesion molecule " relates to the cell adhesion molecule that contains the cell binding sequence.The example of cell adhesion molecule comprises integrin, calcium attachment proteins (cadherin), selects the adhesion molecule of albumen and immunoglobulin superfamily, as VCAM, ICAM, PECAM and NCAM.

Term " ECM adhesion molecule ", " somatomedin " or " cell adhesion molecule " comprise its any active analogue thereof, active fragment or reactive derivative.

About the present invention, term " adhesin polypeptide fragment " is related to cell or influences cell provides the peptide sequence of attachment site in other factor of this lip-deep adhesion, propagation, migration and function, for example improves the peptide sequence of cell attachment efficient.

Several these class adhesin polypeptide fragments are known in the art.Can test segmental binding ability of particular peptide or adhesive capacity according to standard technique.The example of this class peptide sequence includes but not limited to: the peptide sequence that contains RGD; The peptide sequence that contains YIGSR; And/or contain the peptide sequence of IKVAV.

The peptide sequence that contains Arg-Gly-Asp (RGD) is acknowledged as the cell recognition motif.The RGD peptide not only effectively triggers cell adhesion, also can be used for some cell line of selectivity addressing and causes specific cells and reply.Contain the peptide of RGD and the further details of their specific nature about available difference among the present invention and be described in people such as Hersel, among Biomaterials 24 (2003) 4385-4415.

The example of the peptide sequence of the available RGD of containing includes but not limited among the present invention: RGD, RGDS, GRGDS, GRGD, YRGDS, YRGDG, YGRGD, GRGDSP, GRGDSG, GRGDSY, GRGDSPK, CGRGDSY, GCGYGRGDSPG and RGDSP ASSKP.

In a preferred embodiment of the invention, this peptide sequence is Gly-Arg-Gly-Asp-Ser (GRGDS).

The peptide sequence that contains Tyr-Ile-Gly-Ser-Arg (YIGSR) finds on the B1 of laminin chain, and it promotes epithelial cell to adhere to people such as (, Biochemistry, 26, the 6896-900 pages or leaves (1987)) Graf.

The peptide sequence that contains Ile-Lys-Val-Ala-Val (IKVAV) finds on the A of laminin chain, and has been in the news and promotes neurite to grow people such as (, J.Biol.Chem., 264, pp.16174-182 (1989)) Tashiro.

Chemical group of the present invention can comprise repetition peptide sequence (peptide monomer).These repeat peptide sequences can repeat the heteropolymer that homopolymer that peptide monomer forms maybe can be made up of two or more different repetition peptide monomers or subunit by single the kind.Usually, this chemical group can be by 2 to 100 peptide monomers, and common 2 to 50, preferred 3 to 15 compositions.The length of each peptide monomer can be 2 to 40, common 2 to 30, and preferred 2 to 10 amino acid residues.

The technical staff will appreciate that, peptide monomer can chemosynthesis or is equipped with by the genetic recombination length of schooling.Similarly, the chemical group that comprises the repetition peptide sequence can prepare by the peptide monomer chemistry is linked together, or they can be recombinant expressed.

In a specific embodiments of the present invention, chemical group of the present invention comprises the repetition peptide sequence that contains the RGD peptide sequence.

The used in tissue engineering support

Implantable material manufactured according to the present invention can be used for making the used in tissue engineering support.One of advantage of implantable material of the present invention is that it comprises the chemical group of giving the improved biocompatibility of this support.

About the present invention, term " used in tissue engineering support " relates to tissue substituent or implant, for example the functional substitute of (impaired) tissue.

The present invention has special applications widely in humans and animals medical treatment and esthetic surgery, and any and all indications of the used in tissue engineering support of describing before can be used for and be used for expressly open as yet but other the definite easily purposes of those skilled in the art in this area.

The example of the special applications of implantable material of the present invention includes but not limited to: blood vessel (being artificial blood vessel), lymphatic vessel, ureter, trachea, digestive tract, skin, oral cavity, esophagus, stomach wall, urethra and periodontal tissue, cartilaginous tissue and hypodermic substitution material or anatomic implants.Other application is the guard shield of little nerve suture line; With cultured skin carrier (cultured skin carriers).

Support of the present invention is at its each self-application and concrete molding.Different possible shapes include but not limited to hollow pipe, band, cylinder, bar and thin slice.

According to some embodiment, the microbial source cellulose is as the PCM in the support of the present invention.Micro organism cellulose has many favourable character in this respect, and for example it can synthesize different shape or size and have excellent shape retention.These character of micro organism cellulose are mainly owing to the laminar microfibril three dimensional structure of its uniqueness.These microfibril of arranging with nonwoven in form are than thin about 200 times of plant cellulose (as cotton fiber), thereby per unit volume has huge surface area.The method that is used for making molding micro organism cellulose material has description at for example JP 8126697A2, EP 186495, JP 3165774Al and JP 63205109Al.In addition, the manufacturing that is used as the hollow pipe micro organism cellulose of replacement vessels has been described in JP 3272772A2 and EP 396344A2.

Except that implantable material of the present invention, support of the present invention can also comprise auxiliary material.The suitable auxiliary material that are used for this purpose comprise water solublity, dissolve in polymeric material such as agar polar solvent or that form hydrophilic gel, glucosan, polyacrylamide, polyvinyl pyrrolidone, alginate, hyaluronic acid, hot curdlan (curdlan), polyacrylate, heparin, sulfated polysaccharides, amylopectin, carrageenin, glucomannan, cellulose derivative, Polyethylene Glycol, polyvinyl alcohol, gelatin, collagen, laminaritol, Fibronectin, keratin, the silk hydrolyzate, polyamino acid, poly-organic acid and enzyme.By mode implantable material of the present invention and above-mentioned auxiliary material are made up to obtain composite as dipping, lamination or absorption and so on.

According to some embodiment, support of the present invention comprises with the PCM of chemical group derivatization that contains following compositions, and described composition influence contacts with this derivatization PCM or adhesion, growth, migration, propagation, differentiation, shape, polarity and/or the metabolic function of interactional cell.

Therefore, according to the compositions and methods of the invention, can be by the behavior (that is adhesion,, growth, migration, propagation, differentiation, shape, polarity and/or metabolic function) that provides suitable molecule motif to influence any kind cell.

Especially, the chemical group that comprises the composition that influences cell adhesion used according to the invention.

PCM of the present invention can be used for cell adhesion molecule or adhesin polypeptide fragment be and passs various types of cells.The cell of these types comprises any cell that contacts with embedded material in vivo usually.This class cell includes but not limited to epithelial cell, endotheliocyte, fibroblast, sarcoplast, chondroblast, osteoblast and stem cell.Other cell that can be used in method of the present invention and the product comprises Schwann cell, spider cell, oligodendrocyte and their precursor, adrenal pheochromocytoma etc.

The stem cell representative can be expanded easily in cultivation and can be made its offspring class cell of end differentiation eventually by using the particular growth factor.Sarcoplast is the muscle precursor that is obtained by the mesodermal stem cell group at first, for example L-6 and O-CH3 cell.

Recognize that required target cell type can be for example depended in the selection of chemical group used in this invention.But any specific cells adhesion molecule of those skilled in the art's conventional sense or adhesin polypeptide fragment motif are to the adhesive capacity of selected type cell.

In other embodiments, support of the present invention can external pre-inoculating cell, makes cellular exposure thus in this PCM.Can use functional health cell from separate sources (promptly spontaneous, allos or heterogenous cell).The support of these cell inoculations can be used on to be organized in the replacement scenario.According to these embodiments, tissue can external reconstruct, then its implantation is needed among its host.For example, heart sarcoplast can be suspended in the support of the present invention to form the tissue patch of thickness corresponding to heart wall.The heart sticking patch of this reconstruct is implanted as a part of organizing auxotherpy subsequently.Can consider to be used for the similar scheme of blood vessel, cartilage, tendon, bone, skin, nerve and other tissue.

Using method of the present invention, can be that support is in the desired location modification with this implantable material.For example, can be only with a side modification of stent sheet or pipe.The density of PCM and the length of CLM can influence the whole network of PCM or only a side is modified.Therefore, can also change the infiltration of CLM and modification degree and the modification position that the absorption accessibility is optimized PCM thus by the density of change PCM or the length of CLM.According to this embodiment, the invention enables and in the ad-hoc location modification and to give this support/implant dual-use function thus.For example, the inwall of artificial blood vessel can be with the adhesion that promotes human endothelial cell or the chemical group modification of propagation, and the outer wall of same artificial blood vessel can be with promoting the chemical group with the biocompatibility of blood vessel surrounding tissue to come modification.

Support of the present invention can be by the next further modification of pre-cultivation PCM in cell culture medium before seeding cells on this material.Improve the adhesion of cell and this material thus.

In certain embodiments, support of the present invention can be used for preparing artificial blood vessel.The method that acquisition comprises the artificial blood vessel of PCM is as known in the art.Artificial blood vessel of the present invention can have virtually any size, is line style, taper and/or branching.

According to the preferred embodiments of the invention, this artificial blood vessel comprises the microbial source cellulose.EP 0396344 and JP 3272772 have described the method that obtains to comprise the cellulosic artificial blood vessel of microbial source.This micro organism cellulose can be for example produces cellulosic microorganism and obtains by cultivating on the inner surface of the carrier of being made up of for example cellophane, Teflon, silicon, pottery, adhesive-bonded fabric or machine yarn fabric and/or outer surface.

People such as Bodin, Influence of cultivation conditions on mechanicaland morphological properties of bacterial cellulose tubes, BiotechnolBioeng, December in 2006 (electronic publication before publishing) on the 29th has been described and has been obtained to comprise improving one's methods of the cellulosic artificial blood vessel of microbial source.According to this method, by as organic silicone tube top fermentation acetobacter xylinum of oxidation carrier and blast the oxygen of variable concentrations, promptly 21% (air), 35%, 50% and 100% is with form of tubes deposition Bacterial cellulose.

In addition, the method according to this invention is given this micro organism cellulose modification with artificial blood vessel of the chemical group of improved biocompatibility of micro organism cellulose and blood compatibility by bonding as mentioned above.The risk that the improved biocompatibility of artificial blood vessel of the present invention and blood compatibility greatly reduce obstruction and so on.Obstruction is a subject matter, and general under the low relatively flox condition of minor diameter artificial blood vessel.Obstruction is caused by blood coagulation and platelet deposition.Since cause the interaction of these problems to occur in blood vessel-implant at the interface, modification is the mode that improves blood compatibility and reduce obstructing problem according to the present invention.

The feature of artificial blood vessel of the present invention also is high penetration resistance and high burst pressure.

In a preferred embodiment of the invention, the implantable material of this artificial blood vessel comprises and contains the chemical group that at least one contains the peptide sequence of RGD.These sequences promote the adhesion or the propagation of human endothelial cells and blood vessel wall.

In addition, this artificial blood vessel can be inoculated endotheliocyte in advance before implanting.

Tissue displacement and regeneration

The support that comprises the implantable material of the present invention has various medical treatment or operation application.

In accordance with a further aspect of the present invention, provide in-vivo tissue displacement and/or regenerated method, comprised the following steps: a) to provide the used in tissue engineering that comprises implantable material manufactured according to the present invention support; And b) described material is implanted the suitable implantation position of the object that needs it.

About the present invention, term " tissue displacement " and " tissue regeneration " generally refer to organizational project, relate to the replacement of function and the tissue regeneration of damaged tissues.The support that comprises implantable material of the present invention can serve as tissue substituent, thus with the material of implanting substituted host tissue wholly or in part.In addition, the support that comprises the implantable material of the present invention can serve as the tissue regeneration carrier, and host cell permeates this material thus.According to this embodiment, promoted host tissue regeneration.

The method that is used for implanting support of the present invention is those of organizational project that are usually used in for example general surgery, plastic surgery or neurosurgery.These methods adopt according to object and implantation position.Implantation position can be any position of live body, for example vascular system, lymphsystem, skin, nervous system etc. in principle.Object of the present invention can be to organize metathetical any object, for example mammal, preferably people.

In a specific embodiments, before the step of implant frame, with the outer pre-inoculating cell of described stake body.

In another embodiment, the support of used in tissue engineering is an artificial blood vessel.

Provide following embodiment and illustrate the present invention.But, it should be understood that the present invention is intended to be limited to actual conditions and the details of describing among these embodiment.

Embodiment

The inventor uses xyloglucan-GRGDS conjugate with bacterial cellulose surface modification, and the Bacterial cellulose of analysis GRGDS modification is to the adherent influence of endotheliocyte.

Material

In Congo red and xyloglucan absorption research, investigate Bacterial cellulose (BC) hydrogel and Whatmann filter paper (1 grade is made by the textile floss that is used for comparison).In the absorption research of using QCM, use trimethyl silyl cellulose (TMSC).People such as this TMSC such as Kontturi, synthesizing described in Langmuir 19 (2003) 5735-5741.Use Roux flask (100 milliliters of swept volumes), with BC static growth three days in the Semen Maydis pulp fluid medium, produce 2 millimeters pellicles under 30 ℃.Being used for this biosynthetic bacterial strain is acetobacter xylinum subspecies sucrofermentas BPR2001, article number: 1700178 ^TMThis bacterial strain is available from American Type Culture Collection.Under 60 ℃ in 0.1M NaOH, boiled 4 hours, subsequently at Millipore ^TMBoil repeatedly in the water, thereby with the BC purification.This material steam sterilization and cold preservation is standby.Acetone treatment: before lyophilization and sem analysis, spend the night with the acetone treatment Bacterial cellulose.

EXAMPLE l

The preparation of xyloglucan and xyloglucan-GRGDS

Basic pressing in the past at Greffe, L. wait the people, Glycobiology, 2005.15 (4): described in the 437-445 page or leaf, directly handle tamarind core powder (60% xyloglucan content, D.N.Palani, Mumbai by endoglucanase digestion, India), obtain the mixture (15: 7: 32: 46) of XXXG, XLXG, XXLG and XLLG wood ligoglucoside (XGO) thus.Followingly change into corresponding 1-deoxidation-1-aminosuccinamic acid salt derivative (XGO-succ), thus with this XGO activation via 1-deoxidation-1-amino-beta--glucosides.XGO (1 gram, 0.78 mM) is dissolved in the deionized water (10 milliliters), adds ammonium bicarbonate (2.5 gram) then, this mixture was stirred 28 hours down at 42 ℃ in saturated keeping adding ammonium bicarbonate continuously then.By TLC monitoring reaction process (70/30 acetonitrile/water).Remove excessive ammonium bicarbonate by three lyophilization cycle, to produce the white powder of forming by the mixture of XGO and 1-deoxidation-l-amino-beta--glucosides; Based on from the anomeric proton of raw material and product ¹H-NMR signal integration, transforming degree are 83%.This raw product is dissolved in the water (10 milliliters), adds succinic anhydrides (157 milligrams, 1.57 mMs, 2 equivalents), and with this solution vigorous stirring 10 minutes on the vortex mixed machine.With the water that contains 0.1%TFA/acetonitrile mixture anti-phase (C18 silica gel) chromatograph of eluting progressively, produce the XGO-succinamate (860 milligrams, 0.63 mM, two step yields 80%) of white powder. ¹H NMR (500MHz, D ₂O, 25 ℃): δ=2.56 (t, J=6Hz, 2H; COCH ₂CH ₂COOH), 2.61 (t, J=7Hz, 2H; COCH ₂CH ₂COOH), 3.24-3.95 (m; Gal, Glc, the H-2 to H-6 of 1-deoxidation-1-aminosuccinic acid salt-Glc, the H-2 to H-5 of Xyl), 4.44-4.50 (m; The H-1 of Glc and Gal), 4.85-4.89 (m; The H-1 of Xyl), 5.08-5.10 (m; The H-1 that has the Xyl of Gal-(l-2)).ESI-MS[37]: XXXG-succ[M+2Na] ²⁺, 603.6802 value of calculation (603.7095 measured value); XLXG-succ and XXLG-succ[M+2Na] ²⁺, 684.7066 value of calculation (684.7079 measured value); XLLG-succ[M+2Na] ²⁺, 765.7330 value of calculation (765.7475 measured value).

According to people such as Engfeldt, Chembiochem FIELD Full Journal Title:Chembiochem:a European journal of chemical biology, 2005.6 (6): the scheme that the 1043-50 page or leaf is described, use standard solid-phase Fmoc chemistry by the synthetic pentapeptide GRGDS of the scale of 0.25 mM, but following difference is arranged.The aminoacid of in the presence of DIPEA (2.0M in NMP), protecting with HBTU and HOBt (the two all is the 0.45M in DMF) activation Fmoc.Do not carry out termination procedure.In the end after the Fmoc break step, the peptide replacement amount that records this resin is 0.47 mM/gram.In the reactor of being furnished with frit filter (aperture P2), XGO-succ manually is conjugated on the resin-bonded peptide.XGO-succ (260 milligrams, 2 equivalents) is dissolved among the DMF (6 milliliters), and exists down with HBTU (215 milligrams, 6 equivalents) and HOBt (87 milligrams, 6 equivalents) activation at DIPEA (66 milliliters, 4 equivalents).Add resin-bonded peptide (200 milligrams, 1 equivalent) then.After 1 hour, pass through to finish coupling with ethanol, NMP, DIPEA (in DCM, 5%), NMP and DCM (each 10 milliliters) this resin of thorough washing.This resin is dry under vacuum subsequently.At room temperature use 3 milliliters of TFA/H ₂O/TIS (95: 2.5: 2.5) interrupted glycopeptide with 30 minutes from this resin, removed Side chain protective group simultaneously.Water (40 milliliters) dilutes this reaction, extracts with tBME (3 * 40 milliliters), and filters by glass fibre.The lyophilization water produces white solid (82 milligrams, 47% yield).Under the same conditions, from 75 milligrams of resins, unmodified peptide is disconnected and deprotection, produce 15 milligrams of GRGDS (90% yield).

GRGDS: ¹H NMR (500MHz, D ₂O, 25 ℃): δ=1.52-1.81 (m, 4H; 2H ^β-Arg, 2H ^χ-Arg), 2.65 (d, J=6.5Hz, 2H; H ^β-Asp), 3.16 (t, J=7Hz, 2H; H ^δ-Arg), 3.80-3.92 (m, 6H; 4H ^α-Gly, 2H ^β-Ser), 4.26 (t, J=7Hz, 1H; H ^α-Arg), 4.35 (t, J=5Hz, 1H; H ^α-Ser), 4.60 (t, J=6.5Hz, 1H; H ^α-Asp) .ESI-MS:[M+H] ⁺490.2376 value of calculation (490.2109 measured value) XGO-succ-GRGDS: δ=1.53-1.86 (m, 4H; 2H ^β-Arg, 2H ^χ-Arg), 2.55-2.60 (m, 2H; H ^β-Asp), 2.80-2.92 (m, 4H; XGO-NH-COCH ₂CH ₂CO-Gly), 3.12-3.16 (m, 2H; H ^δ-Arg), 3.25-3.98 (m:H ^α-Gly, H ^β-Ser, Gal, Glc, 1-deoxidation-H-2 to H-6 of 1-aminosuccinic acid salt-Glc and the H-2 to H-5 of Xyl), 4.25-4.30 (m, 1H; H ^α-Arg), 4.34-4.36 (m, 1H; H ^α-Ser), 4.68-4.74 (m, the H-1 of Glc and Gal), 4.87-4.91 (m, the H-1 of Xyl), 5.09-5.11 (m has the H-1 of the Xyl of Gal-(l-2)).ESI-MS:XXXG-succ-GRGDS[M+H+Na] ²⁺, 828.2988 value of calculation (828.3080 measured value); XXLG-succ-GRGDS and XLXG-succ-GRGDS[M+H+2Na] ³⁺613.8800 value of calculation (613.8911 measured value), [M+H+Na] ²⁺909.3252 value of calculation (909.3289 measured value); XLLG-succ-GRGDS[M+H+2Na] ³⁺, 667.8976 value of calculation (667.9045 measured value), [M+3Na] ³⁺675.2249 value of calculation (675.2198 measured value), [M+H+Na] ²⁺990.3516 value of calculation (990.3554 measured value).

The following coupling that utilizes the interior glycosyl transferase (XET) of xyloglucan to mediate prepares final xyloglucan-GRGDS glycoconjugate.With the tamarind xyloglucan (Megazyme Ireland) is dissolved in the water (2 mg/ml), and with 200 milliliters with XGO-succ-GRGDS (100 milliliters, at H ₂2 mg/ml among the O), H ₂O (50 milliliters) and PttXET16A enzymatic solution (0.4 units per ml, 50 milliliters, in 100mMNaOAc, pH 5.5) mix.After 35 minutes, by this solution is heated to 85 ℃ 1 hour, stop this reaction.By on glass fiber filter, filtering, remove anaenzyme, and from filtrate, be settled out product by adding ethanol (3 times of volumes).Precipitate is collected on the glass fiber filter, and it is dissolved in the water (20 milliliters) again by stirring and this filter of mild heat.With the lyophilization of gained solution to produce 390 milligrams of XG-GRGDS.The analysis showed that by the HP-SEC in DMSO this product has 32000 M _wValue (M _w/ M _n1.7).Use the same program preparation to have similar molecular weight (M _w36000 (M _w/ M _n1.5)) unmodified xyloglucan, difference is to replace XGO-succ-GRGDS with XGO.

The alternative route of preparation xyloglucan and xyloglucan-GRGDS

With cellulase digestion tamarind core powder TKP (xyloglucan, 1,000,000-1.5 megadaltons of Mw:1), to form low-molecular-weight xyloglucan (XG of low Mw), Mw5000-50000 dalton.Before being connected on the XG that hangs down Mw at the GRGDS peptide, activate the XG (description that sees above) of this low Mw with succinate.This GRGDS peptide is with standard solid-phase Fmoc chemosynthesis, and is connected on the XG of the acidifying low Mw of succinum according to description above.The XG of these GRGDS peptides-low Mw can be directly used in the Bacterial cellulose modification.

Embodiment 2

The absorption of Congo red

(Direct Red 28 is available from Riedel-de by measuring the Congo red dyestuff

Germany) maximal absorptive capacity is evaluated and tested Bacterial cellulose and as the specific surface area of the cotton of reference.Under each adsorption concentration, use No. 1 paper of 6 Whatman and 6 parts of bacterial cellulose gels.This cellulosic material is exposed in the Congo red in the 4 ml water solution that contain 0.5,1.5,2.0,2.5,3.0,3.5,4.0,4.5,5.0 (w/w) Congo red, and the flowing fluid ratio with 100: 1 dyeed 24 hours under 60 ℃.Add NaCl (20%w/w) as electrolyte.Utilize standard curve to absorb the residual concentration [E, mg/ml] that calculates Congo red by the UV under 492 nanometers.Calculate Congo red adsorbance [A, mg/g] on the fiber by the absorption difference of this solution under 492 nanometers before and after the association reaction divided by the fiber quality of every volume solution.

Embodiment 3

The absorption of xyloglucan (XG) and xyloglucan-GRGDS (XG-GRGDS)

Evaluate and test Bacterial cellulose and as the specific surface area of the velveteen of reference by the maximal absorptive capacity of measuring XG and XG-GRGDS.Under each adsorption concentration, use 6 Whatman1 paper and 6 parts of bacterial cellulose gels.This cellulosic material is immersed in the 4 ml water solution that contain 5,10,15,20% (w/w) xyloglucan or xyloglucan-GRGDS.Measure the XG of absorption by the colorimetry (Recueil des Travaux Chimiques desPays-Bas et de 1a Belgique 79 (1960) 675-678) that people such as Kooiman describe.Under each interval of from 0 to 48 hour, extract 200 microlitres, and with 20% (w/v) Na of itself and 5: 1 ₂SO ₄With teriodide solution (0.5%I ₂+ 1%KI) 1 ml soln mixes.Utilize standard curve to calculate the residual concentration [E, mg/ml] of XG by the absorption under 620 nanometers.Calculate the amount [A, mg/g] that is adsorbed on the XG on the fiber by the absorption difference of this solution under 620 nanometers before and after the association reaction divided by the fiber quality of every volume solution.

Embodiment 4

Specific surface area

The formula 1 that utilization is drawn by Langmuir theory of adsorption calculates the velveteen that the absorption by the relative xyloglucan of Congo red and xyloglucan-GRGDS obtains and the specific surface area of Bacterial cellulose:

$\frac{\left[E\right]}{\left[A\right]}=\frac{1}{K_{\mathrm{ads}}{\left[A\right]}_{\max }}+\frac{\left[E\right]}{{\left[A\right]}_{\max }}---\left(1\right)$

[E] concentration (mg/ml) of adsorbate when being adsorption equilibrium wherein, [A] is the amount (milligram/gram cellulose sample) that is adsorbed onto the adsorbate on the cellulose surface, [A _Max] be the maximum (milligram/gram cellulose sample) that is adsorbed onto the adsorbate on the cellulose surface, K _AdsIt is adsorption equilibrium costant.Specific surface area A _SpBe expressed as:

$A_{\mathrm{sp}}=\frac{{\left[A\right]}_{\max }{N}_{\mathrm{<figure-callout\; id="10"\; label="A\; A\; CR"\; filenames="A2008800062120036H1.png,A2008800062120040H1.png"\; state="\{\{state\}\}">A</figure-callout>}}{A}_{\mathrm{CR}}{}_{}}{{10}^{21}\mathrm{Mw}}---\left(2\right)$

Wherein Mw is Congo red (653 a gram/mole); Xyloglucan (36000 gram/mole); The molecular weight of xyloglucan-GRGDS (32000 gram/mole), N _ABe avogadros constant, A _CRBy a Congo red (1.73 square nanometers); Xyloglucan (69 square nanometers); The area that xyloglucan-GRGDS (61 square nanometers) occupies.People such as Ougiya, Bioscience, Biotechnology, and Biochemistry 62 (1998) 1714-1719 calculated the value of Congo red.Descend with molecular weight is linear by hypothesis xyloglucan footprint area, be about to the A of people's such as Ougiya high molecular xyloglucan (980000 gram/mole) _CRThe value of 1870 square nanometers is extrapolated to the xyloglucan of lower molecular weight, i.e. 32000 and 36000 gram/moles obtain the value of xyloglucan and xyloglucan-GRGDS thus.

As shown in Fig. 3 B, obtain straight line by formula (1), show that the absorption of Congo red on these two kinds of substrates all meets Langmuir's model.Therefore the Congo red most probable is adsorbed on these two kinds of cellulose surfaces with form of single sheet.For these two kinds of surfaces, the absorption maximum (A that calculates by slope value _Max) all reach 47mg/g.(72 meters squared per gram) of the specific surface area of BC (79 meters squared per gram) and velveteen about equally, referring to formula (2).The specific surface area of velveteen is reasonably well corresponding to the value that finds in the document about dye adsorption research, and the specific surface area of BC is a little less than the value of previous report.But this species diversity is expected, because BC does not have disintegrate in this case, and extremely may have still less the exposed area that is used to adsorb.

Xyloglucan and xyloglucan-GRGDS also meet the Langmuir absorption behavior, referring to Fig. 4 B and 5B.Absorption maximum (the A of XG and XG-GRGDS _Max) on BC, reach about 180 milligrams/gram, and only be about 3 times of value on velveteen, referring to Fig. 4 A and 5A.The specific surface area of the BC that records with xyloglucan and xyloglucan-GRGDS is about 200 meters squared per gram, and the specific surface area of velveteen is little almost three times, 60 meters squared per gram.These two kinds of cellulose surfaces all meet linear relationship, and promptly large surface area is corresponding to higher xyloglucan adsorbance, referring to Fig. 6.

The difference of xyloglucan adsorbance may be by Bacterial cellulose the swelling network with compare that velveteen is more to be exposed and accessible volume is explained.The size of known adsorbate molecule has appreciable impact to accessible surface area, and this can reach a conclusion: less cotton surface can be used for adsorbing xyloglucan.The Congo red molecule is about 2.5 nanometers along the length of its longitudinal axis, is about 30 nanometers and DP is the xyloglucan skeleton of 26 full extension.The specific surface area difference that is adsorbed onto the xyloglucan on these two kinds of cellulosic substrate also may be because crystalline texture difference.Bacterial cellulose has identical crystal structure with velveteen, i.e. cellulose I, and referring to Fig. 7, and the relative crystallinity of these two kinds of substrates is 70%.But this material has not commensurability crystallization Asia-allomorphism (I α or I β), is 60%I α in BC: 40%I β is 30%I α only: 70%I β in velveteen.This may influence cellulosic physical property, because allomorphism has different crystal accumulations, molecular conformation and hydrogen bonding.

Embodiment 5

Scanning electron microscopy (SEM)

Use SEM to study the configuration of surface of unmodified and modified cellulosic materials.With this bacteria cellulose material in quenching in liquid nitrogen before the lyophilization.Used golden coating surface then before analyzing, this Zeiss DSM 940A that is used in operation under the 10kV carries out.

Embodiment 6

The confocal laser microscopy

The confocal microscopy research Bacterial cellulose that use is furnished with fiber coupling ArKr laser instrument under its wet condition form and the modification in the whole gel.Emission wavelength [λ at dyestuff _Ex=495nm and λ _Em=516] select filter.Should carry out fluorescent labeling with fluorescently-labeled xyloglucan (XG-FITC) by wet Bacterial cellulose sample.Press Brumer, people such as H., Journal of the American Chemical Society, 2004.126 (18): synthetic XG-FITC described in the 5715-5721 page or leaf.With 2 mg/ml XG-FITC liquid storages this wet gel was dyeed 24 hours.After the dyeing,, remove excessive XG-FITC by under gentle agitation, placing deionized water to spend the night in sample.

Embodiment 7

ESCA

With before the xyloglucan surface modification and afterwards, measure the chemical composition of Bacterial cellulose with ESCA.After modification and before measuring, this material is dried down at 30 ℃.Use the Quantum 2000 of Physical Electronics to measure.Analyzed area is 500 * 500 square microns, and beam sizes is 100 microns.Angle between sample and the detector is 45 °.Measure peak intensity, and use the MultiPak software of Physical Electronics to carry out curve fitting.Cellulosic feature ESCA spectrum have 286.7eV corresponding to peak of the carbon that closes with the oxygen singly-bound and 287.9eV corresponding to a peak of the carbon of two oxygen bondings.Calculate the relative quantity of different bonded carbons with the gaussian curve approximation at highest resolution C1s peak.The diverse location of C-C, C-O, O-C-O or C=O and O-C=O is respectively 285.0 ± 0.2eV, 286.7 ± 0.2eV, 281.1 ± 0.2eV and 289.4 ± 0.2eV.

ESCA shows that the xyloglucan modification has been used on this surface.The carbon amount on the oxygen that is bonded to the xyloglucan side group of can seeing increases slightly.But, may be because this group size be little and may embed when oven dry and point to gel inside, so can't quantize the amount of GRGDS.Also there is trace nitrogen xyloglucan and cellulose in itself, and this makes sign further complicated.

Embodiment 8

Dynamic contact angle with water

Carrying out static contact angle on the cellulose membrane of 6 oven dry (unmodified and with the cellulose of XG or XG-GRGDS modification) measures.On each cellulose surface, apply 5 microlitre drops.Use clinometer by be recorded in this solid and and the tangent line on drop surface between the angle that forms measure contact angle θ _e

The unmodified Bacterial cellulose of comparing, during with xyloglucan and xyloglucan-GRGDS modification, wettability slightly high (table 1).

Table 1

The surface	Contact angle, θ e±SD
		Bacterial cellulose	?44±5.3
Bacterial cellulose+XG	?29±4.8
		Bacterial cellulose+XG-GRGDS	?32±5.8

The contact angle of unmodified Bacterial cellulose and water is relative to be greatly because consolidated structure, less hole (for capillary force) and less available hydroxyl (owing to high-crystallinity).Increase available hydroxyl with the xyloglucan modification, and improved wettability thus.Introduce GRGDS and can significantly not reduce wettability, referring to the structure among Fig. 8.Wettability still is higher than unmodified BC.

Embodiment 9

Use the protein adsorption of QCM

Use QCM-D instrument (Q-sense AB,

Sweden) absorption of research protein on cellulose surface is subjected to the influence of surface modification.

The plain surface of preparation model fibre on gold-plated QCM-D crystal.The surface was cleaned 10 minutes in the UV/ ozone chamber, then at Milli-Q water, H ₂O ₂(30%) and NH ₃(25%) flooded 10 minutes down in 70 ℃ in 5: 1: 1 mixture.This surface is with the Milli-Q washing and use nitrogen drying.With trimethyl silyl cellulose (in toluene, 1 mg/ml) with 4000rpm, be spun on the gold surface in 1 minute.Remove trimethyl silyl, and go up the generation cellulose at hydrogen chloride steam (10% solution).Under third harmonic (15Hz), measure.The variation reflection of f is coupled to the quality on the plane of crystal.For thin equally distributed stiffness films, be associated with the quality absorption as the frequency displacement of absorption being brought out as described in the Sauerbrey formula (Δ f):

$\frac{m}{A}=\frac{\mathrm{Cf}}{n_r}$

Wherein m is quality (ng), and A is area (cm ²), n _rBe that (=1,3...) C is mass sensitivity constant (17.7ng/cm to harmonic number ²/ Hz).Measure in triplicate.Absorption xyloglucan and xyloglucan-GRGDS under 2 mg/ml concentration.After introducing cell culture medium, the step of after each absorption, carrying out the water desorbing.Use with cell inoculation in used identical culture medium research protein adsorption.This cell culture medium contains protein mixture, comprises the cell adhesion protein Fibronectin that contains the RGD motif.In order to illustrate the increase that whether causes adsorbed proteins from cell culture medium (particularly Fibronectin) with the XG-GRGDS modification, use QCM-D on the plain surface of model fibre, to adsorb research.(whether Fibronectin antibody (Biotin) Abcam), is Fibronectin to confirm any protein that may be adsorbed (ab6584) to introduce Fibronectin antibody after introducing cell culture medium.The step of after all absorption, carrying out the water desorbing.

As shown in Figure 9, about 100 nanogram/square centimeters are adsorbed on the unmodified cellulose surface from the protein of cell culture medium.When adsorbing xyloglucan in the ban, there is not protein to be adsorbed.When with having the xyloglucan that adheres to pentapeptide when cellulose modified, compare with unmodified cellulose, still less protein is adsorbed (50 nanogram/square centimeter).After with xyloglucan-GRGDS and cell culture medium modification, introduce the Fibronectin IgG antibody.The proteic IgG of tissue adhesion does not adsorb, and shows that adsorbed protein is not the attachment proteins Fibronectin or is not activated form at least.

Embodiment 10

Radix Rumicis X-ray scattering (WAXS)

The lyophilization pill of BC is pressed into the pill of 1 centimetre of diameter.On the SiemensD5000 diffractometer, write down x-ray diffraction pattern.Use wavelength

CuK α anode.Scan by 2 θ=5-30 °.Measure the intensity at the crystal diffraction peak of amorphous diffraction.Relative crystallinity is determined as the ratio between crystal block section and the general headquarters' branch.

Embodiment 11

Cell inoculation

Use enzymatic method to isolate endotheliocyte (HSVEC) from the saphenous healthy position of people.(PAA Laboratories GmbH, Linz cultivate in Austria) cell, and this M199 is supplemented with and contains the 1.7-3.4g/dl albumin and total protein content is the 20% hyclone (FBS of 3-4.5g/ml at M199; PAA Laboratories GmbH), penicillin-streptomycin (100U/mL; PAA Laboratories GmbH), 1.2mM L-glutaminate (PAA), Medulla Bovis seu Bubali extract (75mg/500mL; Make in the laboratory) and heparin (13U/mL; LeoPharma,

And hold it under 37 ℃ and contain 5%CO Sweden), ₂The humidification incubator in.Before the cell bed board, this BC modification is spent the night with 15% xyloglucan and the XG-GRGDS that are equivalent to the BC dry weight.Before the cell bed board, modified cellulose is washed twice with PBS.

In order to assess cellular morphology, with HSVEC with 3 * 10 ⁵The density bed board of individual cells/square cm is on modification and unmodified BC plate.Taking out sample at the 1st day and the 3rd day evaluates and tests.With cell fixation saturatingization processing in 3.7% formaldehyde and in 0.2%Triton X-100.In order to make the f-actin as seen, with being attached to Alexa

546 (MolecularProbes Inc., Eugene, OR, USA) phalloidin on is with cell dyeing.With nucleus DAPI (Sigma-Aldrich Sweden AB, Stockholm, Sweden) counterstain.Sample is contained in Slowfade ^TMIn the Antifade mounting medium (Molecular Probes Inc.) and with Axio Imager M1 (Carl Zeiss,

Germany) analyze.Carry out digitally captured with AxioCam HRc (Zeiss) to picture.

Cell adhesion

Initial cell adheres to and studies show that, cell adhesion is faster and better on the cellulose of unmodified and xyloglucan modification at ratio on the cellulose of xyloglucan-GRGDS modification.Optical microscopic image shows, has more many cells on modified surface, and stretch and adhere to more flourishing, referring to Figure 10.

Embodiment 12

Form

The form of velveteen and Bacterial cellulose is different in many aspects.Velveteen by its surface by microfibril cover fibrous.Fiber size is about 6 microns.On the other hand, Bacterial cellulose is by the fibrillated swelling three-dimensional network of the nanometer that is of a size of the 70-100 nanometer.

Can not change this form (Figure 11 C) at aqueous phase with the xyloglucan modified bacteria cellulose.If carry out modification then situation is really not so in organic solvent (for example acetone), this moment, network obviously shrank comparison diagram 11A and 11B.In order to keep the network of BC, preferably modification in water.(xyloglucan-FITC) the burnt Z-scanning of the copolymerization of the Bacterial cellulose of modification shows the even modification of the whole quilt of this nanofiber cellulosic material with the fluorescence xyloglucan.

Conclusion

The present inventor has described under the impregnable situation of form of nanometer fibril network the new method with the modification of cellulose nanometer fibril.As confirming, use xyloglucan-GRGDS with Bacterial cellulose success modification with colorimetry.Adsorbance reaches maximum 190 milligrams/gram.As by SEM with the z-scanning in the confocal microscopy arrives, the whole material of nanofiber cellulosic material is by evenly modification.In addition, in organic solvent, obviously help keeping form at the aqueous phase modification ratio.This modification improves wettability, is adsorbed that protein reduces or amount can be ignored shown in this possible explanation QCM-D.Initial cell research is verified, and when BC hydrogel during with xyloglucan-GRGDS modification, the adhesion of endotheliocyte improves.Confirm that as QCM-D improved cell adhesion is not owing to non-specific adsorption Fibronectin from culture medium, but owing to presenting the RGD epi-position because of the specificity of XG.

Although describe specific embodiments of the present invention in detail by embodiment, those skilled in the art obviously can expect the present invention is made amendment and adjusts.But, understand clearly that this class is revised and is adjusted in as claimed in claim the scope of the invention.

Claims (33)

1. by polymer carbohydrate material (PCM) modification being prepared the method for implantable material, described modification is attached on the PCM by making the carbohydrate link molecule (CLM) that comprises chemical group, and wherein said chemical group is given described PCM improved biocompatibility.

2. according to the process of claim 1 wherein the described method preparation of CLM that comprises chemical group: by xyloglucan polymer manufacture xyloglucan fragment by comprising the following steps; With one or more chemical groups are connected on segmental reducing end of described xyloglucan and/or the side chain, make thus and be applicable to the CLM that comprises chemical group that is attached on the described PCM.

3. utilize the following step to carry out the step of described PCM modification according to the process of claim 1 wherein:

(a) provide the carbohydrate polymer fragment (CPF) that comprises chemical group;

(b) the described CPF that comprises chemical group and solubility polymerization carbohydrate (SCP) are contacted under the condition that causes forming the complex of being made up of described CPF that comprises chemical group and SCP, described CPF and SCP form carbohydrate link molecule (CLM) together; With

(c) described CLM and the PCM that wants modification in that being attached under the condition on the described PCM, described CLM are contacted.

4. according to the method for the implantable material of preparation of claim 1, its by in conjunction with xyloglucan source molecule with the cellulosic material modification, on the molecule of described xyloglucan source, be connected with the chemical group of giving the improved biocompatibility of described cellulosic material.

5. according to the method for the implantable material of preparation of claim 4, described method is by carrying out the cellulosic material modification, and comprises the following steps:

(a) provide the xyloglucan-oligosaccharide that has been connected with the chemical group of giving improved biocompatibility on it;

(b) described xyloglucan-oligosaccharide and the carbohydrate polymer that comes from xyloglucan are contacted causing forming under the condition of carbohydrate link molecule (CLM), described link molecule comprises the xyloglucan-oligosaccharide of chemical group with the connection and comes from the carbohydrate polymer of xyloglucan; With

(c) described CLM and the cellulosic material of wanting modification are contacted making CLM be attached on the cellulosic material and improve under the condition of biocompatibility of cellulosic material.

6. according to the method for claim 4-5, the wherein said chemical group of giving improved biocompatibility is protein or peptide.

7. according to the method for claim 4-6, wherein make covalently bound chemical group be connected to the reducing end of xyloglucan-oligosaccharide.

8. according to the method for claim 5, the formation of wherein said CLM is by glycosyl transferase (XET, EC 2.4.1.207) catalysis in the xyloglucan.

9. according to the method for claim 3, wherein said PCM is a cellulosic material, described SCP comes from xyloglucan, and described CPF comes from xyloglucan and contains 3-100 polymer backbone monosaccharide unit, and described chemical group is the factor of giving improved biocompatibility.

10. according to the method for claim 9, the wherein said CPF that comes from xyloglucan contains 4-10 polymer backbone monosaccharide unit.

11., wherein make the covalently bound reducing end of described chemical group to CPF according to the method for claim 10-11.

12., the CLM that comprises chemical group is contacted and bonded step is carried out under aqueous conditions with PCM according to each method among the claim 1-11.

13. according to each method among claim 3,5-7 or the 9-12, the CPF that comprises described chemical group is contacted in the presence of the enzyme with glycosyl transfer activity with SCP, and described endonuclease capable promotes to form the complex of being made up of at least a portion of described CPF that comprises chemical group and SCP.

14. according to the method for claim 13, wherein said enzyme with glycosyl transfer activity is a glycosyl transferase (XET, EC 2.4.1.207) in the xyloglucan.

15. according to each method among claim 3 or the 12-14, wherein said CPF is xyloglucan-oligosaccharide (XGO), at least a portion of described SCP comes from xyloglucan (XG).

16. according to each method among the claim 1-15, wherein said PCM is the cellulosic material form.

17. according to each method among the claim 1-16, wherein said PCM is the microbial source cellulose, preferred described microbial source cellulose is generated by acetobacter xylinum (Acetobacterxylinum).

18. according to each method among the claim 1-17, the chemical group of the improved biocompatibility of the wherein said PCM of giving comprises following at least a: extracellular matrix (ECM) adhesion molecule, somatomedin, cell adhesion molecule or adhesin polypeptide fragment.

19. according to each method among the claim 1-18, the chemical group of the improved biocompatibility of the wherein said PCM of giving comprises anticoagulin.

20. according to the method for claim 18, wherein said adhesin polypeptide fragment is: the peptide sequence that contains Arg-Gly-Asp (RGD); The peptide sequence that contains Tyr-Ile-Gly-Ser-Arg (YIGSR); And/or contain the peptide sequence of Ile-Lys-Val-Ala-Val (IKVAV).

21. according to the method for claim 20, wherein said chemical group comprises at least one peptide sequence that contains RGD.

22. according to the method for claim 21, wherein said chemical group comprises at least one Gly-Arg-Gly-Asp-Ser (GRGDS) peptide sequence.

23. according to each method among the claim 1-22 make the medical treatment or the operation usefulness implantable material.

24. the implantable material of making according to each method among the claim 1-22 is used to make the purposes of used in tissue engineering support.

25. according to the purposes of claim 24, it is used to make artificial blood vessel, artificial skin, neural support or orthopaedic implants.

26. according to the purposes of claim 24, it is used to make artificial blood vessel.

27. comprise the used in tissue engineering support of the material of making according to each method among the claim 1-22.

28. according to the support of claim 27, the external pre-inoculating cell of wherein said used in tissue engineering support.

29. comprise the artificial blood vessel of the material of making according to each method among the claim 1-22.

30. according to the artificial blood vessel of claim 29, the external pre-inoculating cell of wherein said artificial blood vessel.

31. in-vivo tissue displacement and/or regenerated method comprise the following steps:

A) provide the used in tissue engineering support that comprises according to each implantable material of making among the claim 1-22; With

B) described material is implanted the suitable implantation position of the object that needs it.

32. according to the method for claim 31, wherein before the step of implanting described used in tissue engineering support, with the outer pre-inoculating cell of described stake body.

33. according to each method among the claim 31-32, wherein said used in tissue engineering support is an artificial blood vessel.

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