CN101668552B - Implantable material comprising cellulose and glycopeptides xyloglucan-GRGDS - 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

Implantable material comprising cellulose and the glycopeptide xyloglucan-GRGDS

technical field

The present invention relates to implantable materials for medical or surgical use, comprising specific chemical groups that modify the physicochemical properties of said material in order to impart suitable implantable or biocompatible properties to it. More specifically, the present invention relates to implantable materials comprising polymeric carbohydrates, methods of making these implantable materials and the use of materials made by these methods and products comprising these materials.

Background technique

Organ or tissue failure is a serious health problem. Tissue engineering offers the potential to recreate tissue function using functional healthy cells from different sources (ie, autologous, allogenic or xenogeneic cells) and/or extracellular natural or synthetic polymers.

A large number of synthetic polymer materials with various properties are used today in medical and cosmetic applications such as prostheses, implants and scaffolds for tissue engineering. These synthetic polymer materials can generally be divided into two main categories: temporary implants/bioresorbable and long-term implants/non-bioresorbable. Examples of bioresorbable synthetic materials include polymers comprising poly-l-lactic acid (PLLA) and poly-l-glycolic acid (PLGA). An example of a long-term implantable and non-bioresorbable material is poly(tetrafluoroethylene)PTFE, which has been used in a variety of medical implantable articles, including vascular grafts and tissue repair sheets and patches.

However, these synthetic materials also have limitations and disadvantages such as inappropriate interactions between polymers and cells causing foreign body reactions in vivo such as inflammation, infection, aseptic loosening, local tissue waste and implant cysts, and thrombus Formation and embolism. Thus, the success of promising polymers depends in part on the attachment and growth of relevant cells on their surfaces. Surface chemistry mediates the cellular response to the material and affects cell adhesion, proliferation, migration and function on the surface.

In the field of arterial revascularization there is an increasing need for functional small diameter artificial grafts (inner diameter < 6 mm). When self-replacement of the blood vessel is not available (eg, due to the poor condition of the patient's vasculature), the surgeon has no choice but to implant a synthetic polymer-based blood vessel. After implantation, the initial major problem with these vessels is the almost immediate occlusion caused by blood clotting and platelet deposition under relatively low flow conditions. Since the interactions leading to surface-induced thrombosis occur at the blood-biomaterial interface, surface modification has been a way to improve hemocompatibility. Among different modifications, modification with polysaccharides such as heparin has also been widely used to minimize thrombus formation on artificial organs. However, the problem is that when heparin is directly bound to the surface, the activity of heparin is significantly reduced. To date, the most successful type of biocompatible surface is the end-point-attached heparin surface.

Another attractive surface modification is the introduction of hydrophilic groups that prevent plasma protein adsorption, platelet adhesion, and thrombus formation. The problem is keeping them on the surface of long-term devices such as vascular grafts. In particular, water-soluble polymers such as polyacrylamide (PAAm); poly(N,N-dimethylacrylamide) (PDMAAm); poly(ethylene glycol) (PEG), ethylene-vinyl alcohol copolymer ( EVA) and poly(2-hydroxyethyl methacrylate) (PHEMA) were grafted onto solid surfaces to prevent protein adsorption.

Other concepts for modifying materials to improve their hemocompatibility have to try to mimic biologically inert surfaces. In blood vessels, this inert surface consists of a single layer of endothelial cells. Materials currently used as vascular grafts, such as expanded polytetrafluoroethylene (ePTFE) and polyester, do not promote human endothelial cell adhesion or proliferation. Surfaces are therefore treated with fibrinogen, fibrinogen or with immobilized RGD (Arg-Gly-Asp), which is the smallest fragment of the active site of adhesion proteins such as fibrinogen, fibrinogen, and von Willbrand factor. modified. In addition to surface modification with heparin, extensive research and trials have been conducted to seed endothelial cells onto PTFE tubing. But the problem is keeping the cells on the surface for long periods of time.

However, these synthetic materials mentioned above, such as PTFE and polyester, also have other limitations and disadvantages, including a limited range of physical and biochemical properties. Therefore, there is still a need to search for alternative implantable materials that are more suitable for specific surgical applications.

Several investigators have studied the tissue biocompatibility of cellulose and its derivatives and examined some specific applications of this material. In particular, the use of cellulose produced by microorganisms in tissue engineering has been investigated. Microbial-derived cellulose has a network structure in which very fine ribbon-like fibers composed of highly crystalline and highly uniaxially oriented cellulose are intricately entangled with each other, and this network structure contains a large amount of liquid in its internal voids. Since the cellulose is composed of many ribbon-shaped fibers with high crystallinity, the cellulose can resist external force such as stretching force even in a wet state. Microbial cellulose is not structurally different from plant-derived cellulose, but a highly ordered structure such as the above-mentioned structure is not found in plant-derived cellulose, which is characteristic of microbial cellulose. Correspondingly, microbial cellulose has high strength despite being gel-like.

US 6800753 describes the use of regenerated cellulose (RC) and oxidized regenerated cellulose (ORC) for the preparation of scaffolds for tissue engineering. RC and ORC composites were prepared by first dissolving cellulose in a solvent system and then regenerating the cellulose into the desired scaffold structure. To fabricate porous scaffolds, porogens are introduced into the solvent system to create pores in the scaffold structure. The scaffold can then be oxidized to introduce carboxyl, aldehyde and/or ketone functional groups on its surface. These functional groups serve as sites for cell adhesion or further chemical modification to induce cell adhesion and subsequent proliferation.

Seo S et al. ("Alginate microcapsules prepared with xyloglucanas a synthetic extracellular matrix for hepatocyte attachment", Biomaterials, Vol. 26 no.17 (2005), pp. 3607-3615) describe the modification with xyloglucan (XG) Calcium alginate polymeric carbohydrate capsules to prepare a synthetic extracellular matrix of primary mouse hepatocytes. The improved liver-specific function was attributed to the specific interaction between the galactose moiety of XG and the asialoglycoprotein receptor on hepatocytes.

Yang Y et al. ("Biodegradable scaffolds-delivery systems forcell therapies", Expert Opinion on Biological Therapy volume 6, no.5 (2006), pages 485-498) discuss the use of biomolecules to modify the surface of biodegradable materials review article. Biodegradable scaffolds are said to be important in facilitating the application of cell therapy. Scaffold material selection is discussed, especially with regard to biocompatibility. The formation of biomimetic scaffolds, novel fabrication techniques capable of controlling scaffold architecture and microstructure, and fabrication of injectable and in situ crosslinked scaffolds are outlined. It is outlined that challenges remain in providing biodegradable scaffolds that can properly guide the cells they come into contact with.

Zhou Q et al. ("Xyloglucan and xyloglucan endotransglycosy-lases (XET): Tools for ex vivo cellulose surface modification", Biocatalysis and Biotransformation, volume 24, no.1-2 (2006), pages 107-120) are wood A review article in the field of fiber technology describing the modification of wood fibers to provide novel biomaterials and novel surface modification methods for cellulose. Provides a new system for attaching functional groups to wood pulp, including high-affinity interactions between xyloglucan and cellulose, intraxyloglucan glycosyltransferases catalyzed polysaccharide-oligosaccharide coupling reactions Combination of unique properties and traditional carbohydrate synthesis.

However, challenges remain in providing implantable materials with good biocompatibility, especially with surfaces comprising bioactive factors.

There are various problems with the use of regenerated cellulose (RC) and oxidized regenerated cellulose (ORC) made according to US 6800753. For example, treating cellulose with a solvent may not be ideal because such treatment can be complicated by structural changes in the cellulosic material. These structural changes lead to shrinkage and altered morphology as well as to more brittle materials.

Currently, as mentioned above, implantable materials suffer from problems related to their physical and biochemical properties, such as their surface chemistry. There is no doubt that there is currently a long-standing but unmet need to develop new implantable materials and improve their properties so that they can be successfully used in medical applications.

Accordingly, the main object of the present invention is to provide an improved method for preparing implantable materials having excellent compatibility with a living body, and to provide implantable materials produced by this method.

Another object of the present invention is to provide implantable materials with desirable mechanical properties such as mechanical and tensile strength, elongation and suturability.

Another object of the present invention is to provide implantable materials with sites for cell attachment or other factors affecting cell adhesion, proliferation, migration and function.

Yet another object of the present invention is to provide implantable materials suitable for implantation in vivo.

The means and means of accomplishing each of the above objects, as well as others, will become apparent from the following description of the invention.

Contents of the invention

In order to achieve the object of the present invention, the present inventors provide a new method for preparing implantable materials, comprising modifying the PCM by bonding a carbohydrate linking molecule (CLM) comprising a chemical group to a polymeric carbohydrate material (PCM). wherein the chemical group imparts improved biocompatibility to the PCM.

One of the main advantages of the method of the present invention for the preparation of implantable materials comprising PCM modified with chemical groups is the avoidance of chemical treatment of the PCM. This treatment changes the confirmation or orientation and other physicochemical properties of the PCM. Thus, the method of the present invention avoids the loss of fiber structure and properties commonly encountered in the direct chemical modification of PCMs.

For example, chemical modification of hydrogels such as bacterial cellulose is complicated by structural changes due to the use of organic solvents. The present invention avoids these structural changes by applying aqueous chemical-enzymatic techniques to the surface modification of bacterial cellulose.

In other aspects of the present invention, there are provided implantable materials made according to the method of the present invention; the use of the implantable material for the manufacture of scaffolds for tissue engineering; tissue engineering materials comprising materials made according to the present invention scaffolds; and methods of tissue replacement and/or regeneration in vivo.

According to a specific embodiment, the present invention includes an artificial blood vessel comprising an implantable material made according to the present invention. The artificial blood vessel of the present invention is characterized by high penetration resistance, high burst pressure and good biocompatibility.

The invention is described in more detail below, especially with reference to the accompanying drawings.

Description of drawings

Figure 1 shows an unmodified polymeric carbohydrate material (PCM) (1) and a modified PCM (6). The modified PCM comprises a carbohydrate linking molecule (CLM) (2) comprising at least a portion of a soluble polymeric carbohydrate (SCP) (3) and a chemical group (5) and optionally complexed group of carbohydrate polymer fragments (CPF) (4).

Figure 2 shows the preparation of CLM (2) using enzyme and CPF (4). The SCP (8) is contacted with an enzyme (7) and a CPF (4) containing a chemical group (5). The enzyme (7) cleaves the SCP and introduces the chemical group-containing CPF to give the product CLM (2).

Figure 3 shows the Langmuir adsorption isotherms (A) of bacterial cellulose ▲ and cotton linters stained with Direct Red 28 (Congo Red). The lines in panel B represent the results of linear regression.

Figure 4 shows the Langmuir adsorption isotherms (A) of bacterial cellulose adsorbed with xyloglucan· and xyloglucan-GRGDS▲. The lines in panel B represent the results of linear regression.

Figure 5 shows the Langmuir adsorption isotherms (A) of cotton linters adsorbed with xyloglucan· and xyloglucan-GRGDS▲. The lines in panel B represent the results of linear regression.

Figure 6 shows the adsorption amount of xyloglucan ● and xyloglucan-GRGDS▲ as a function of the specific surface area of the cellulose substrate. The line represents the result of linear regression.

Figure 7 shows the crystallinity of bacterial cellulose, cotton linters and lyocell.

Figure 8 shows the chemical structure of GRGDS xyloglucan oligosaccharide (XGO-GRGDS).

Figure 9 shows adsorption of cell culture medium to cellulose (-), adsorption of xyloglucan and then cell culture medium to cellulose (...), adsorption of xyloglucan-GRGDS and then cell culture medium to cellulose The QCM adsorption isotherm of (—··). Arrows represent water washes.

Figure 10 shows the optical visualization of ECs of unmodified bacterial cellulose (A), xyloglucan-modified bacterial cellulose (B) and xyloglucan-GRGDS modified bacterial cellulose (C). micro image.

Figure 11 shows SEM images of untreated bacterial cellulose (A), bacterial cellulose treated in acetone (B) and bacterial cellulose modified with xyloglucan-GRGDS (C).

Detailed ways

The present invention relates to the development of implantable polymeric carbohydrate materials (PCM) for medical or surgical use, comprising specific chemical groups on their surface to modify the physicochemical properties of said material. In particular, said chemical groups confer improved biocompatibility on the PCM, for example by providing attachment sites for cells or other factors affecting cell adhesion, proliferation, migration and function on the surface.

Furthermore, the invention relates to methods for the preparation of the implantable materials of the invention as well as to the use of the materials produced by these methods, and to products comprising these materials.

In this specification, unless otherwise specified, "a" or "an" means "one or more".

In relation to the present invention, the term "biocompatibility" relates to the property of a material, ie the property of a material to be compatible with a living body. In other words, in harmony with living organisms; without toxic or impairing effects on biological functions. A material has poor compatibility with a living body if, for example, an adverse reaction is induced when the material is brought into contact with a part of the living body. This adverse reaction can cause foreign body reactions such as inflammation, infection, aseptic loosening, local tissue waste and implant cysts, as well as thrombosis and embolism. On the contrary, if such adverse reactions do not occur, good compatibility with the living body is observed. Furthermore, improved biocompatibility means improved compatibility of the material with living organisms. An example of biocompatibility is hemocompatibility, the property of a material to be compatible with blood.

Methods of making implantable materials

According to a first aspect of the present invention, there is provided a method of modifying a polymeric carbohydrate material (PCM) to prepare an implantable material by bonding a carbohydrate linking molecule (CLM) comprising a chemical group to the PCM, wherein The chemical groups endow the PCM with improved biocompatibility.

One embodiment of this method is shown in Figure 1, which shows an unmodified PCM (1) and a carbohydrate linking molecule (CLM) (2) comprising at least a portion of an SCP (3) and A chemical group (5) and an optionally complexed carbohydrate polymer fragment (CPF) (4) comprising the chemical group. Since the CLM is capable of bonding to the PCM, bonding occurs when the PCM is brought into contact with the CLM.

In one embodiment of the invention, the method comprises the steps of: (a) providing a carbohydrate polymer fragment (CPF) comprising a chemical group; (b) combining said CPF comprising the chemical group with a soluble polymeric carbohydrate a compound (SCP) is contacted under conditions that result in the formation of a complex consisting of said CPF comprising the chemical group and the SCP, said CPF and SCP together forming a carbohydrate linking molecule (CLM); and (c) causing said The complex is contacted with the PCM to be modified under conditions such that the complex is bound to the PCM.

In a preferred embodiment, the step of contacting and bonding the CLM comprising chemical groups to the PCM is performed under aqueous conditions. This has the advantage that the PCM morphology is not altered during the step of contacting and bonding the CLM to the PCM.

The method of preparing a CLM comprising a chemical group, ie the method of preparing a complex consisting of said CLM and said chemical group is carried out separately from PCM. Thus, the method for preparing CLMs containing chemical groups can be scaled up and includes several steps and harsh conditions.

The term "polymeric carbohydrate material" abbreviated "PCM" relates to a material comprising a water-insoluble polymeric carbohydrate material and/or a water-soluble polymeric carbohydrate material which consists in whole or in part of repeating units of one or more monosaccharides composition. Such PCMs are usually composite materials containing two or more different types of polymeric carbohydrates or a carbohydrate-containing polymer and another polymer such as a protein.

According to the present invention, PCM may be any polymeric carbohydrate material suitable for use as an implantable material, for example as a main component of a scaffold for tissue engineering. Different PCMs usable in the present invention are for example described in WO 03/033813.

In a particular embodiment, the PCM is in the form of a cellulosic material, ie comprises cellulose. In the present invention, cellulose can be extracted from annual plants (such as flax, hemp or corn) or perennial plants (such as cotton, poplar, birch, willow, eucalyptus, larch wood, pine or spruce). Examples of suitable cellulosic materials include purified cotton, cotton linters, alpha-cellulose, wood pulp, virgin wood pulp, powdered cellulose, microcrystalline cellulose, and/or cellulose modified to other polymers.

Another source of cellulose that has different properties compared to plant cellulose is microbial cellulose. Cellulose of microbial origin has attracted attention for use as a biomaterial, mainly due to the possibility of molding it into different shapes for a given application, as well as its biocompatibility and high purity. This cellulose is an exopolysaccharide and is produced relatively cheaply by culturing the bacterium Acetobacter xylinum. In contrast to plant cellulose, this cellulose is extruded in its pure form and not bound to any other polymers or proteins. Bacterial cellulose can be efficiently purified with sodium hydroxide to achieve the FDA endotoxin value for grafts in contact with blood, which is <20 EU per device. Bacterial cellulose contains 99% water and is considered (though not by definition) a hydrogel. Despite its low solids content, the branched network of nanocellulose fibrils provides good mechanics.

The term "microbial cellulose" relates to cellulose produced by microorganisms, such as bacteria, as described above. In a preferred embodiment of the invention, a culture of cellulose-synthesizing bacteria such as Acetobacter xylinum is used.

The term "soluble carbohydrate polymer" abbreviated (SCP) relates to polymers comprising one or more different monosaccharides or derivatives thereof, which are soluble in aqueous or organic solvents. Examples include those carbohydrate polymers classified as hemicelluloses (not just those composed of β(1-4)-linked glucose units, i.e. cellulose), pectins (polyuronic acids and esters) and starches (with or without Polysaccharides of α(1-4)-linked polydextrose) containing α(1-6) side chain branches. Xyloglucan is a polysaccharide composed of a β(1-4)-linked polyglucose backbone with α(1-6) xylose residues, which themselves can be further Sugars such as fucose and arabinose are substituted; it is an example of this type of SCP, specifically hemicellulose. In a preferred embodiment, the SCP is capable of binding to the PCM, for example via one or more hydrogen bonds, ionic interactions, one or more covalent bonds, van der Waals forces, or any combination of these. In one embodiment of the invention, the SCP may be a CPF according to the description below. In a preferred embodiment, the SCP is derived from xyloglucan (XG).

"Derived from xyloglucan" refers to the product with α(1-6) xylose residues (the α(1-6) xylose residues themselves can be further replaced by other sugars such as fucose and arabinose) Polysaccharides composed of β(1-4)-linked polyglucose backbones, and their further chemically substituted and modified variants and fragments.

The term "carbohydrate polymer fragment" abbreviated "CPF" relates to molecules that may be enzymatically or chemically produced fragments of SCP. Examples of such fragments include any number of repeat units of the SCP.

Thus suitable fragments may contain from 2 to about 5000 monosaccharide units in the polymer backbone, such 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, 3001-4000 or 4001-5000 monosaccharide units. The CPF may further comprise side chains of different lengths and compositions. Specific examples include, but are not limited to, xylogluco-oligosaccharides (XGO) or fragments thereof, or further modified with one or more fucose residues or other monosaccharides.

XGO is generally named according to the nomenclature system outlined in Fry et al. (1993) Physiologia Plantarum, 89, 1-3, where G represents an unsubstituted β-glucopyranosyl residue and X represents xylopyranosyl-α (1 -6) Glucopyranosyl unit, L represents galactopyranosyl-β(1-2)-xylopyranosyl-α(1-6)glucosyl unit, F represents fucopyranosyl- α(1-2)-galactopyranosyl-β(1-2)-xylopyranosyl-α(1-6)-glucosyl unit. These different units can be linked between glucopyranosyl units via β(1-4) linkages to form a β(1-4)-glucan polysaccharide backbone. Using this nomenclature, the XGOs typically isolated after endoglucanase digestion of tamarind xyloglucan are XXXG, XLXG, 25XXLG and XLLG. If the reducing end glucose (G) of these oligosaccharides is in the reduced sugar alcohol form, the unit is represented by "Gol". Thus, reduced (sugar alcohol) derivatives of the above oligosaccharides, for example from tamarind xyloglucan, are designated XXXGol, XLXGol, XXLGol and XLLGol.

Preferably, the CPF is derived from xyloglucan and may contain from 3 to 100, including 4 to 10, polymer backbone monosaccharide units.

The term "carbohydrate-linked molecule" abbreviated "CLM" relates to a molecule or complex containing at least a part and a chemical group of an SCP according to the above description. The CLM can be bonded to the PCM, eg, via one or more hydrogen bonds, ionic interactions, one or more covalent bonds, van der Waals forces, or any combination of these.

CLMs can be prepared by organic or chemical synthesis and/or by exploiting the catalytic activity of certain enzymes.

In one embodiment of the present invention, CLM can be prepared using the method described in WO 2004/094646A1. For example, the CLM comprising chemical groups can be prepared by a method comprising the steps of: preparing a xyloglucan fragment from a xyloglucan polymer; and attaching one or more chemical groups to the xyloglucan fragment On the reducing end and/or on the side chain, thereby making a CLM containing chemical groups that can be used for bonding to PCM.

An embodiment of CLM preparation using enzymes and CPF is shown in FIG. 2 . The SCP (8) is contacted with an enzyme (7) and a CPF (4) containing a chemical group (5). In this embodiment, the enzyme (7) cleaves the SCP to introduce the chemical group-containing CPF, resulting in the product CLM (2).

The CLM may contain one or more chemical groups.

In one embodiment of the invention, the CLM can be prepared using enzymes capable of transferring native or chemically modified mono- or oligosaccharides onto the ends of oligo- or polysaccharides. Such enzymes include, but are not limited to, enzymes with high transglycosylation activity but low hydrolytic activity, glucosylhydrolases with high intrinsic transglycosylation activity, enzymes that have been engineered to increase their transglycosylation activity, and Glucosyltransferases that use nucleotide sugars as substrates.

The different enzymes that can be used according to the invention and their properties and how to obtain them are described in more detail in WO 03033813.

In a preferred embodiment of the invention, the enzyme is xyloglucan endoglycosyltransferase (XET, EC 2.4.1.207).

In nature, glycosyltransferases such as XET enzymes function in living plants, so the enzyme is clearly capable of working in an aqueous environment. The method of the present invention can thus be carried out in aqueous solution, or it can be carried out in water with certain components such as buffers and/or wetting agents and/or stabilizers and/or polymers and/or organic components which reduce water activity (such as DMSO) in the presence of implementation. See WO 03/033813 for further details on these components.

According to a specific embodiment of the present invention, the new XGO can be synthesized by first coupling chemically modified XGO to xyloglucan (XG) using XET enzyme in solution, and then allowing the modified XG to adsorb onto the cellulosic material. Chemical groups are added to cellulosic materials that do not contain intrinsic xyloglucan.

In the present invention, the term "chemical group" refers to any chemical group potentially useful for PCM modification. Modification refers to changing the functional properties of PCM. The ability to alter the functional properties of PCM is in this sense inherent in this chemical group. According to the invention, this modification confers improved biocompatibility on the PCM. Therefore, this chemical group modifies the physicochemical properties of the PCM to make it more biocompatible.

Biocompatibility refers to the property of a material to be compatible with living organisms. One way to improve the biocompatibility of a material is to affect the adhesion, development, migration, proliferation, differentiation, shape, polarity and/or metabolic function of cells in contact with or interacting with the material. One particular way of improving the biocompatibility of a material is to reduce the tendency of the material to induce clotting of blood that comes into contact with the material. This anticoagulant property is especially useful for materials used in artificial blood vessels.

Components contained in the chemical groups of the present invention that alter the physicochemical properties of PCM to make it more biocompatible include, but are not limited to: anticoagulant factors, ECM adhesion molecules, growth factors, cell adhesion molecules and adhesion Attached peptide fragments as well as cell culture substrates and cellular nutrients. It will be clear to those skilled in the art that this list of ingredients is not exhaustive and other suitable ingredients that alter the physicochemical properties of PCM to make it more biocompatible can be used in the present invention.

In the present invention, the term "anticoagulant factor" relates to molecules that reduce the tendency of blood to clot. Preferred examples of such molecules for use in the present invention are heparins, low molecular weight heparins and pentasaccharide inhibitors of factor Xa, such as fondaparinux and idraparinux.

In relation to the present invention, the term "ECM adhesion molecule" relates to extracellular macromolecules that make up the extracellular matrix (ECM). These macromolecules (mainly proteins and polysaccharides) are secreted locally and assemble into organized 3-D networks in the extracellular space of most tissues. ECM molecules include glycosaminoglycans and proteoglycans such as chondroitin sulfate, fibronectin, heparan sulfate, hyaluronidase (hyaluron), dermatan sulfate, keratin sulfate, laminin, collagen, heparan sulfate protein Glycans and elastin. The extracellular matrix regulates the organization of the intracellular cytoskeleton, cell differentiation, and the spatial organization of cells and tissues. Indeed, the ECM plays a key role in regulating the behavior of cells in contact with it by affecting the development, migration, proliferation, differentiation, shape, polarity and metabolic functions of that cell.

In relation to the present invention, the term "growth factor" relates to biologically active polypeptides that cause cell proliferation. They include but are not limited to epidermal growth factor, transforming growth factor, nerve growth factor, acidic and basic fibroblast growth factor and angiogenic factors, platelet-derived growth factor, insulin and insulin-like growth factors including somatomodulin, myxoma and Vaccinia virus derived growth factor.

In relation to the present invention, the term "cell adhesion molecule" relates to a cell adhesion molecule comprising a cell binding sequence. Examples of cell adhesion molecules include integrins, cadherins, selectins, and adhesion molecules of the immunoglobulin superfamily, such as VCAM, ICAM, PECAM, and NCAM.

The term "ECM adhesion molecule", "growth factor" or "cell adhesion molecule" includes any active analogue, active fragment or active derivative thereof.

In relation to the present invention, the term "adhesion peptide fragment" relates to peptide sequences that provide attachment sites for cells or other factors affecting cell adhesion, proliferation, migration and function on the surface, eg peptide sequences that improve cell attachment efficiency.

Several such adhesion peptide fragments are known in the art. Specific peptide fragments can be tested for their ability to bind or adhere according to standard techniques. Examples of such peptide sequences include, but are not limited to: RGD-containing peptide sequences; YIGSR-containing peptide sequences; and/or IKVAV-containing peptide sequences.

Peptide sequences containing Arg-Gly-Asp (RGD) are recognized as cellular recognition motifs. RGD peptides are not only effective in triggering cell adhesion, but can also be used to selectively address certain cell lines and elicit specific cellular responses. Further details on the different RGD-containing peptides useful in the present invention and their specific properties are described in Hersel et al., Biomaterials 24 (2003) 4385-4415.

Examples of RGD-containing peptide sequences useful in the present invention include, but are not limited to: RGD, RGDS, GRGDS, GRGD, YRGDS, YRGDG, YGRGD, GRGDSP, GRGDSG, GRGDSY, GRGDSPK, CGRGDSY, GCGYGRGDSPG, and RGDSP ASSKP.

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

A peptide sequence containing Tyr-Ile-Gly-Ser-Arg (YIGSR) is found on the B1 chain of laminin, which promotes epithelial cell attachment (Graf et al., Biochemistry, 26, pp. 6896-900 (1987)).

A peptide sequence containing Ile-Lys-Val-Ala-Val (IKVAV) is found on the A chain of laminin and has been reported to promote neurite outgrowth (Tashiro et al., J. Biol. Chem., 264, pp. .16174-182(1989)).

The chemical groups of the invention may comprise repeating peptide sequences (peptide monomers). These repeat peptide sequences may be homopolymers composed of a single repeat peptide monomer or may be heteropolymers composed of two or more different repeat peptide monomers or subunits. Typically, the chemical group may consist of 2 to 100 peptide monomers, usually 2 to 50, preferably 3 to 15. Each peptide monomer may be 2 to 40, usually 2 to 30, preferably 2 to 10 amino acid residues in length.

The skilled artisan will recognize that peptide monomers can be chemically synthesized or prepared by recombinant genetics. Similarly, chemical groups comprising repetitive peptide sequences can be prepared by chemically linking together peptide monomers, or they can be expressed recombinantly.

In a particular embodiment of the invention, the chemical group of the invention comprises a repeating peptide sequence comprising an RGD peptide sequence.

Scaffolds for Tissue Engineering

Implantable materials made according to the present invention can be used to manufacture scaffolds for tissue engineering. One of the advantages of the implantable material of the present invention is that it contains chemical groups that endow the scaffold with improved biocompatibility.

In relation to the present invention, the term "scaffold for tissue engineering" relates to tissue substitutes or implants, eg functional substitutes for (damaged) tissue.

The present invention has wide specific application in human and animal medical and cosmetic surgery, and can be used for any and all indications of scaffolds for tissue engineering as previously described, and for applications not expressly disclosed in the art but readily ascertainable by those skilled in the art. other uses.

Examples of specific applications of the implantable materials of the present invention include, but are not limited to: blood vessels (i.e., artificial blood vessels), lymphatic vessels, ureters, trachea, digestive tract, skin, oral cavity, esophagus, abdominal wall, urethra, and periodontal tissue, cartilage tissue and subcutaneous tissue replacement materials or tissue implants. Other applications are shielding for microneural sutures; and cultured skin carriers.

The stents of the present invention are specifically shaped for their respective applications. Different possible shapes include, but are not limited to, hollow tubes, strips, cylinders, rods and sheets.

According to certain embodiments, microbially derived cellulose is used as PCM in the scaffolds of the invention. Microbial cellulose has many advantageous properties in this regard, such as its ability to be synthesized in various shapes or sizes and its excellent shape retention. These properties of microbial cellulose are mainly attributed to its unique three-dimensional structure of sheet-like microfibrils. Arranged in a nonwoven form, these microfibrils are approximately 200 times thinner than plant cellulose, such as cotton fibers, and thus have an enormous surface area per unit volume. Methods for making shaped microbial cellulose materials are described in, for example, JP 8126697A2, EP 186495, JP 3165774A1 and JP 63205109A1. Furthermore, in JP 3272772A2 and EP 396344A2 the manufacture of hollow tube microbial cellulose for use as a blood vessel substitute is described.

In addition to the implantable material of the invention, the stent of the invention may also contain auxiliary materials. Suitable auxiliary materials for this purpose include water-soluble, polar solvent-soluble or hydrophilic gel-forming polymeric materials such as agar, dextran, polyacrylamide, polyvinylpyrrolidone, alginate, Hyaluronic acid, curdlan, polyacrylate, heparin, sulfated polysaccharide, pullulan, carrageenan, glucomannan, cellulose derivatives, polyethylene glycol, polyvinyl alcohol , gelatin, collagen, laminbutol, fibronectin, keratin, silk hydrolysates, polyamino acids, polyorganic acids and enzymes. The implantable material of the present invention is combined with the aforementioned auxiliary materials by means such as impregnation, lamination or adsorption to obtain a composite material.

According to certain embodiments, the scaffolds of the invention comprise PCM derivatized with chemical groups comprising components that affect the adhesion, development, migration, proliferation, Differentiation, shape, polarity and/or metabolic function.

Thus, according to the compositions and methods of the present invention, the behavior (i.e., adhesion, development, migration, proliferation, differentiation, shape, polarity and/or metabolic function) of any type of cell can be influenced by providing appropriate molecular motifs .

In particular, chemical groups comprising components affecting cell adhesion are used according to the invention.

The PCMs of the present invention can be used to present cell adhesion molecules or adhesion peptide fragments to various types of cells. These types of cells include any cells that are normally in contact with implant materials in vivo. Such cells include, but are not limited to, epithelial cells, endothelial cells, fibroblasts, myoblasts, chondrocytes, osteoblasts, and stem cells. Other cells that may be used in the methods and products of the invention include Schwann cells, astrocytes, oligodendrocytes and their precursors, adrenal chromaffin cells, and the like.

Stem cells represent a class of cells that can be readily expanded in culture and whose progeny can be terminally differentiated by the administration of specific growth factors. Myoblasts are muscle precursor cells originally derived from mesoderm stem cell populations, such as L-6 and O-CH3 cells.

It will be appreciated that the choice of chemical moieties for use in the present invention may depend, for example, on the desired target cell type. Those skilled in the art can routinely test the ability of any particular cell adhesion molecule or adhesion peptide fragment motif to adhere to a cell type of choice.

In other embodiments, scaffolds of the invention can be pre-seeded with cells in vitro, thereby exposing the cells to the PCM. Functional healthy cells from different sources (ie autogenous, allogenic or xenogenic cells) can be used. These cell-seeded scaffolds can be used in tissue replacement protocols. According to these embodiments, tissue can be reconstituted in vitro and then implanted into a host in need thereof. For example, cardiomyoblasts can be suspended in the scaffolds of the invention to form a patch of tissue with a thickness corresponding to the wall of the heart. This reconstituted heart patch is then implanted as part of tissue replacement therapy. Similar protocols can be considered for blood vessels, cartilage, tendon, bone, skin, nerves and other tissues.

Using the methods of the present invention, the implantable material, the scaffold, can be modified at desired locations. For example, only one side of the scaffold sheet or tube may be modified. The density of the PCM as well as the length of the CLM can affect whether the entire network of the PCM or just one side is modified. Therefore, the degree of modification and the location of modification of PCM can be optimized by changing the density of PCM or the length of CLM and thus the permeation and adsorption accessibility of CLM. According to this embodiment, the invention makes it possible to modify at specific positions and thus give the scaffold/implant a dual function. For example, the inner wall of an artificial blood vessel can be modified with chemical groups that promote adhesion or proliferation of human endothelial cells, while the outer wall of the same artificial blood vessel can be modified with chemical groups that promote biocompatibility with perivascular tissue.

Scaffolds of the invention can be further modified by pre-cultivating the PCM in cell culture medium prior to seeding cells onto the material. The adhesion of cells to the material is thereby improved.

In certain embodiments, the stents of the present invention can be used to prepare artificial blood vessels. Methods of obtaining artificial blood vessels comprising PCM are well known in the art. The prosthetic vessels of the present invention can be of any size, linear, tapered and/or branched.

According to a preferred embodiment of the invention, the artificial blood vessel comprises cellulose of microbial origin. EP 0396344 and JP 3272772 describe methods for obtaining artificial blood vessels comprising cellulose of microbial origin. The microbial cellulose can be obtained, for example, by culturing cellulose-producing microorganisms on the inner and/or outer surfaces of a support consisting of, for example, cellophane, Teflon, silicon, ceramic, nonwoven or woven fabric.

Bodin et al., Influence of cultivation conditions on mechanical and morphological properties of bacterial cellulose tubes, Biotechnol Bioeng, 29 December 2006 (epub ahead of publication) describe an improved method for obtaining artificial blood vessels comprising microbially derived cellulose. According to this method, bacterial cellulose was deposited in the form of tubes by fermenting Acetobacter xylinum on silicone tubes as oxidizing supports and bubbling with different concentrations of oxygen, namely 21% (air), 35%, 50% and 100%.

Furthermore, according to the method of the present invention, the microbial cellulose of the artificial blood vessel is modified by bonding chemical groups that impart improved biocompatibility and hemocompatibility to the microbial cellulose as described above. The improved biocompatibility and hemocompatibility of the artificial blood vessel of the present invention greatly reduces the risk of occlusion and the like. Occlusion is a major problem and is prevalent under relatively low flow conditions in small diameter grafts. The blockage is caused by blood clotting and platelet deposition. Since the interactions responsible for these problems occur at the vessel-implant interface, modification according to the present invention is a way to improve hemocompatibility and reduce occlusion problems.

The vascular prosthesis of the present invention is also characterized by a high penetration resistance and a high burst pressure.

In a preferred embodiment of the invention, the implantable material of the artificial blood vessel comprises a chemical group comprising at least one RGD-containing peptide sequence. These sequences promote the adhesion or proliferation of human endothelial cells to the vessel wall.

In addition, the artificial blood vessel can be pre-seeded with endothelial cells prior to implantation.

tissue replacement and regeneration

Stents comprising implantable materials of the present invention have various medical or surgical applications.

According to another aspect of the present invention, there is provided a method for tissue replacement and/or regeneration in vivo, comprising the steps of: a) providing a scaffold for tissue engineering comprising an implantable material made according to the present invention; and b) incorporating said Implantation of a material requires a proper implantation site in its subject.

In relation to the present invention, the terms "tissue replacement" and "tissue regeneration" refer generally to tissue engineering, involving the functional replacement and regeneration of damaged tissue. Scaffolds comprising implantable materials of the invention can act as tissue substitutes, whereby host tissue is completely or partially replaced with the implanted material. In addition, scaffolds comprising implantable materials of the invention may serve as tissue regeneration vehicles whereby host cells infiltrate the material. According to this embodiment, host tissue regeneration is promoted.

Methods for implanting the inventive scaffolds are those commonly used in tissue engineering, eg in general surgery, orthopedic surgery or neurosurgery. These methods are employed depending on the subject and the implantation location. In principle, the implantation site can be any part of the living body, such as vascular system, lymphatic system, skin, nervous system, etc. The subject of the present invention may be any subject in need of tissue replacement, such as a mammal, preferably a human.

In a specific embodiment, prior to the step of implanting the scaffold, the scaffold is pre-seeded with cells in vitro.

In another embodiment, the scaffold for tissue engineering is an artificial blood vessel.

The following examples are given to illustrate the invention. It should be understood, however, that the invention is not intended to be limited to the specific conditions and details described in these examples.

Example

The present inventors modified the surface of bacterial cellulose with xyloglucan-GRGDS conjugates, and analyzed the effect of GRGDS-modified bacterial cellulose on endothelial cell adhesion.

Material

Bacterial cellulose (BC) hydrogel and Whatmann filter paper (grade 1, made of pure cotton linter for comparison) were investigated in Congo red and xyloglucan adsorption studies. Trimethylsilylcellulose (TMSC) was used in adsorption studies using QCM. The TMSC was synthesized as described in Kontturi et al., Langmuir 19 (2003) 5735-5741. Using Roux flasks (100 mL working volume), BC were statically grown in corn steep liquor for three days at 30°C to produce a 2 mm pellicle. The strain used for this biosynthesis was Acetobacter xylinum subsp. sucrofermentas BPR2001, product number: 1700178 ^™ . This strain was purchased from American Type Culture Collection. BC was purified by boiling in 0.1M NaOH at 60°C for 4 hours, followed by repeated boiling in Millipore ^(TM) water. The material is steam sterilized and refrigerated until ready to use. Acetone treatment: BC was treated with acetone overnight prior to freeze-drying and SEM analysis.

Example 1

Preparation of xyloglucan and xyloglucan-GRGDS

Tamarind kernel flour (60% xyloglucan content , DN Palani, Mumbai, India), thus obtaining a mixture of XXXG, XLXG, XXLG and XLLG xyloglucan oligosaccharides (XGO) (15:7:32:46). The XGO was activated via conversion of 1-deoxy-1-amino-β-glycoside to the corresponding 1-deoxy-1-aminosuccinamic acid salt derivative (XGO-succ) as follows. XGO (1 g, 0.78 mmol) was dissolved in deionized water (10 mL), then ammonium bicarbonate (2.5 g) was added, and the mixture was then incubated at 42 °C while continuously adding ammonium bicarbonate to maintain saturation. Stir for 28 hours. The progress of the reaction was monitored by TLC (70/30 acetonitrile/water). Excess ammonium bicarbonate was removed by three freeze-drying cycles to yield a white powder consisting of a mixture of XGO and 1-deoxy-l-amino-β-glycoside; ¹ H-NMR based on anomeric protons from starting material and product Integrating the signal, the degree of conversion was 83%. The crude product was dissolved in water (10 mL), succinic anhydride (157 mg, 1.57 mmol, 2 equiv) was added, and the solution was stirred vigorously on a vortex mixer for 10 minutes. Reverse phase (C18 silica gel) chromatography, stepwise eluted with a water/acetonitrile mixture containing 0.1% TFA, gave XGO-succinamic acid salt (860 mg, 0.63 mmol, 80% yield over two steps) as a white powder. ¹ H NMR (500MHz, D ₂ O, 25°C): δ = 2.56 (t, J = 6Hz, 2H; COCH ₂ CH ₂ COOH), 2.61 (t, J = 7Hz, 2H; COCH ₂ CH ₂ COOH), 3.24-3.95 (m; Gal, Glc, H-2 to H-6 of 1-deoxy-1-aminosuccinate-Glc, H-2 to H-5 of Xyl), 4.44-4.50 (m; Glc and H-1 of Gal), 4.85-4.89 (m; H-1 of Xyl), 5.08-5.10 (m; H-1 of Xyl with Gal-□(1-2)). ESI-MS[37]: XXXG-succ[M+2Na] ²⁺ , 603.6802 calculated (603.7095 observed); XLXG-succ and XXLG-succ[M+2Na] ²⁺ , 684.7066 calculated (684.7079 observed) ; XLLG-succ[M+2Na] ²⁺ , calcd. 765.7330 (765.7475 found).

The pentapeptide GRGDS was synthesized on a 0.25 millimolar scale using standard solid-phase Fmoc chemistry according to the protocol described by Engfeldt et al., Chembiochem FIELD Full Journal Title: Chembiochem: a European journal of chemical biology, 2005.6(6): pp. 1043-50 , but with the following differences. Fmoc protected amino acids were activated with HBTU and HOBt (both 0.45M in DMF) in the presence of DIPEA (2.0M in NMP). No capping step was performed. After the final Fmoc cleavage step, the resin was measured to have a peptide substitution of 0.47 mmol/g. XGO-succ was manually conjugated to the resin-bound peptide in a reactor equipped with a frit filter (pore size P2). XGO-succ (260 mg, 2 eq) was dissolved in DMF (6 mL) and treated with HBTU (215 mg, 6 eq) and HOBt (87 mg, 6 eq) in the presence of DIPEA (66 mL, 4 eq). activation. The resin-bound peptide (200 mg, 1 equiv) was then added. Coupling was terminated after 1 h by washing the resin extensively with ethanol, NMP, DIPEA (5% in DCM), NMP and DCM (10 mL each). The resin was then dried under vacuum. Glycopeptides were interrupted from the resin using 3 mL of TFA/ _H2O /TIS (95:2.5:2.5) at room temperature for 30 minutes while removing side chain protecting groups. The reaction was diluted with water (40 mL), extracted with tBME (3 x 40 mL), and filtered through glass fiber. The aqueous phase was lyophilized to yield a white solid (82 mg, 47% yield). The unmodified peptide was cleaved and deprotected from 75 mg of resin under the same conditions to yield 15 mg of GRGDS (90% yield).

GRGDS: ¹ H NMR (500MHz, D ₂ O, 25°C): 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; ^Hδ -Arg), 3.80-3.92(m, 6H; ^4Hα -Gly, ^2Hβ- Ser), 4.26(t, J=7Hz, 1H; H ^α -Arg), 4.35(t, J=5Hz, 1H; Hα ^- Ser), 4.60(t, J=6.5Hz, 1H; ^Hα -Asp).ESI-MS: [M+H] ⁺ 490.2376 calculated Value (490.2109 found values) 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 ₂ CH ₂ CO-Gly), 3.12-3.16 (m, 2H; H ^δ -Arg), 3.25-3.98 (m: H ^α -Gly, H ^β -Ser, Gal, Glc, 1-deoxy-1-aminosuccinate-H-2 to H-6 of Glc and 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, H-1 of Glc and Gal), 4.87-4.91 (m, H-1 of Xyl), 5.09-5.11 (m, with Gal- □(1-2) of Xyl of H-1). ESI-MS: XXXG-succ-GRGDS[M+H+Na] ²⁺ , calculated at 828.2988 (828.3080 measured); XXLG-succ-GRGDS and XLXG-succ-GRGDS[M+H+2Na] ³⁺ calculated at 613.8800 Value (613.8911 observed value), [M+H+Na] ²⁺ 909.3252 calculated value (909.3289 observed value); XLLG-succ-GRGDS[M+H+2Na] ³⁺ , 667.8976 calculated value (667.9045 observed value), [ M+3Na] ³⁺ 675.2249 calcd (675.2198 found), [M+H+Na] ²⁺ 990.3516 calcd (990.3554 found).

The final xyloglucan-GRGDS glycoconjugate was prepared using xyloglucan endoglycosyltransferase (XET) mediated coupling as follows. Dissolve tamarind xyloglucan (Megazyme, Ireland) in water (2 mg/ml), and mix 200 ml with XGO-succ-GRGDS (100 ml, 2 mg/ml in _H2O ), _H2O O (50 mL) and PttXET16A enzyme solution (0.4 units/mL, 50 mL in 100 mM NaOAc, pH 5.5) were mixed. After 35 minutes, the reaction was terminated by heating the solution to 85°C for 1 hour. The denatured enzyme was removed by filtration on a glass fiber filter and the product was precipitated from the filtrate by adding ethanol (3 volumes). The precipitate was collected on a glass fiber filter and redissolved in water (20 mL) by stirring and gently heating the filter. The resulting solution was lyophilized to yield 390 mg of XG-GRGDS. Analysis by HP-SEC in DMSO showed that the product had a _Mw value of 32000 ( _Mw / _Mn 1.7). Unmodified xyloglucan with similar molecular weight ( _Mw 36000 ( _Mw / _Mn 1.5)) was prepared using the same procedure, except that XGO-succ-GRGDS was replaced by XGO.

Alternative pathways for the preparation of xyloglucan and xyloglucan-GRGDS

Digest tamarind kernel powder TKP (xyloglucan, Mw: 1 million-1.5 million Daltons) with cellulase to form low molecular weight xyloglucan (XG with low Mw), Mw 5000-50000 lanes Leon. The low Mw XG was activated with succinate prior to attachment of the GRGDS peptide to the low Mw XG (see description above). The GRGDS peptide was synthesized using standard solid phase Fmoc chemistry and linked to succinylated low Mw XG as described above. These GRGDS peptides-XG with low Mw can be directly used to modify bacterial cellulose.

Example 2

Adsorption of Congo Red

Germany)最大吸附量来评测细菌纤维素和作为参比的棉的比表面积。在各吸附浓度下使用6张Whatman 1号纸和6份细菌纤维素凝胶。将该纤维素材料在含0.5、1.5、2.0、2.5、3.0、3.5、4.0、4.5、5.0(w/w)刚果红的4毫升水溶液中暴露于刚果红中，并在60℃下以100∶1的液体比染色24小时。加入NaCl(20％w/w)作为电解质。利用标准曲线由492纳米下的UV吸收来计算刚果红的残留浓度[E，毫克/毫升]。由结合反应前后该溶液在492纳米下的吸附差值除以每体积溶液的纤维质量来计算纤维上的刚果红吸附量[A，mg/g]。By measuring Congo red dye (Direct Red 28, purchased from Riedel-de

Germany) maximum adsorption capacity to evaluate the specific surface area of bacterial cellulose and cotton as a reference. Six sheets of Whatman No. 1 paper and six bacterial cellulose gels were used at each adsorption concentration. The cellulosic material was exposed to Congo red in 4 ml of aqueous solution containing 0.5, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 (w/w) Congo red, and at 60° C. at 100: 1 liquid ratio to stain for 24 hours. NaCl (20% w/w) was added as electrolyte. The residual concentration of Congo red [E, mg/ml] was calculated from the UV absorbance at 492 nm using the standard curve. The adsorption amount [A, mg/g] of Congo red on the fiber was calculated by dividing the adsorption difference of the solution at 492 nm before and after the binding reaction by the fiber mass per volume of the solution.

Example 3

Adsorption of xyloglucan (XG) and xyloglucan-GRGDS (XG-GRGDS)

The specific surface area of bacterial cellulose and cotton linter as a reference was evaluated by determining the maximum adsorption capacity of XG and XG-GRGDS. Six sheets of Whatman No. 1 paper and six bacterial cellulose gels were used at each adsorption concentration. The cellulosic material was soaked in 4 ml of aqueous solution containing 5, 10, 15, 20% (w/w) xyloglucan or xyloglucan-GRGDS. Adsorbed XG was measured by the colorimetric method described by Kooiman et al. (Recueil des Travaux Chimiques desPays-Bas et de la Belgique 79 (1960) 675-678). Withdraw 200 μl at various time intervals from 0 to 48 hours and mix it with a 5:1 solution of 20% (w/v) _Na2SO4 and _triiodide (0.5% _I2 + 1% KI) 1 ml of the solution was mixed. The residual concentration of XG [E, mg/ml] was calculated from the absorbance at 620 nm using the standard curve. The amount [A, mg/g] of XG adsorbed on the fiber was calculated by dividing the adsorption difference at 620 nm of the solution before and after the binding reaction by the fiber mass per volume of the solution.

Example 4

specific surface area

The specific surface area of lint and bacterial cellulose obtained by the adsorption of Congo red to xyloglucan and xyloglucan-GRGDS was calculated using Equation 1 derived from the Langmuir adsorption theory:

$\frac{<span\; class="notranslate">\; [</span>\mathrm{<span\; class="notranslate">\; E.</span>}<span\; class="notranslate">\; ]</span>}{<span\; class="notranslate">\; [</span>\mathrm{<span\; class="notranslate">\; A</span>}<span\; class="notranslate">\; ]</span>}<span\; class="notranslate">\; =</span>\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{{\mathrm{<span\; class="notranslate">\; K</span>}}_{\mathrm{<span\; class="notranslate">\; ads</span>}}{<span\; class="notranslate">\; [</span>\mathrm{<span\; class="notranslate">\; A</span>}<span\; class="notranslate">\; ]</span>}_{\mathrm{<span\; class="notranslate">\; max</span>}}}<span\; class="notranslate">\; +</span>\frac{<span\; class="notranslate">\; [</span>\mathrm{<span\; class="notranslate">\; E.</span>}<span\; class="notranslate">\; ]</span>}{{<span\; class="notranslate">\; [</span>\mathrm{<span\; class="notranslate">\; A</span>}<span\; class="notranslate">\; ]</span>}_{\mathrm{<span\; class="notranslate">\; max</span>}}}<span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 1</span>}<span\; class="notranslate">\; )</span>$

where [E] is the concentration of adsorbate at adsorption equilibrium (mg/ml), [A] is the amount of adsorbate adsorbed onto the cellulose surface (mg/g cellulose sample), and [A _max ] is the adsorption The maximum amount of adsorbate (mg/g cellulose sample) onto the cellulose surface, K _ads is the adsorption equilibrium constant. The specific surface area A _sp is expressed as:

${\mathrm{<span\; class="notranslate">\; A</span>}}_{\mathrm{<span\; class="notranslate">\; sp</span>}}<span\; class="notranslate">\; =</span>\frac{{<span\; class="notranslate">\; [</span>\mathrm{<span\; class="notranslate">\; A</span>}<span\; class="notranslate">\; ]</span>}_{\mathrm{<span\; class="notranslate">\; max</span>}}{\mathrm{<span\; class="notranslate">\; N</span>}}_{\mathrm{<span\; class="notranslate">\; <figure-callout\; id="10"\; label="A\; A\; CR"\; filenames="H2008800062123E00021.png,H2008800062123E00061.png"\; state="\{\{state\}\}">A</figure-callout></span><figure-callout\; id="10"\; label="A\; A\; CR"\; filenames="H2008800062123E00021.png,H2008800062123E00061.png"\; state="\{\{state\}\}">\; </figure-callout>}}{\mathrm{<span\; class="notranslate">\; A</span>}}_{}{\mathrm{<span\; class="notranslate">\; CR</span>}}_{}}{{\mathrm{<span\; class="notranslate">\; 10</span>}}^{\mathrm{<span\; class="notranslate">\; twenty\; one</span>}}\mathrm{<span\; class="notranslate">\; mw</span>}}<span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 2</span>}<span\; class="notranslate">\; )</span>$

where Mw is the molecular weight of Congo red (653 g/mol); xyloglucan (36000 g/mol); xyloglucan-GRGDS (32000 g/mol), N _A is Avogadro's constant, A _CR is the area occupied by one Congo red (1.73 nm2); xyloglucan (69 nm2); xyloglucan-GRGDS (61 nm2). Congo red values were calculated by Ougiya et al., Bioscience, Biotechnology, and Biochemistry 62 (1998) 1714-1719. By assuming that the area occupied by xyloglucan decreases linearly with molecular weight, i.e. extrapolating the value of _ACR 1870 nm2 for high molecular weight xyloglucan (980,000 g/mol) of Ougiya et al. to lower molecular weight xyloglucan, That is 32000 and 36000 g/mol, from which the values for xyloglucan and xyloglucan-GRGDS were obtained.

As shown in Fig. 3B, a straight line was obtained from equation (1), indicating that the adsorption of Congo red on both substrates fit the Langmuir model. Congo red was therefore most likely adsorbed on these two cellulose surfaces in the form of a monolayer. The adsorption maximum (A _max ) calculated from the slope values reached 47 mg/g for both surfaces. The specific surface area of BC (79 m2/g) is approximately equal to that of cotton linters (72 m2/g), see equation (2). The specific surface area of lint corresponds fairly well to the values found in the literature on dye adsorption studies, whereas that of BC is slightly lower than that reported earlier. However, this difference is expected since BC did not disintegrate in this case and most likely has less exposed area for adsorption.

Xyloglucan and xyloglucan-GRGDS also conformed to the Langmuir adsorption behavior, see Figures 4B and 5B. The adsorption maximum (A _max ) of XG and XG-GRGDS reached about 180 mg/g on BC and was only about 3 times the value on cotton linters, see Figures 4A and 5A. The specific surface area of BC measured with xyloglucan and xyloglucan-GRGDS was approximately 200 m2/g and that of cotton linters was almost three times smaller at 60 m2/g. Both cellulose surfaces follow a linear relationship, ie a larger surface area corresponds to a higher xyloglucan adsorption capacity, see Figure 6.

The difference in xyloglucan adsorption may be explained by the swollen network of BC and the greater exposed and accessible volume compared to cotton linters. The size of the adsorbate molecules is known to have a significant effect on the accessible surface area, which leads to the conclusion that less cotton surface is available for adsorption of xyloglucan. The length of the Congo red molecule along its longitudinal axis is about 2.5 nanometers, while the fully extended xyloglucan backbone with a DP of 26 is about 30 nanometers. The difference in the specific surface area of xyloglucan adsorbed to these two cellulosic substrates may also be due to the difference in crystalline structure. Bacterial cellulose and cotton linters have the same crystal structure, cellulose I, see Figure 7, and the relative crystallinity of both substrates is 70%. However, this material has varying amounts of crystalline sub-alpha (Iα or Iβ), from 60% Iα: 40% Iβ in BC to only 30% Iα: 70% Iβ in cotton linters. This may affect the physical properties of cellulose, as allomorphs have different crystal packing, molecular conformation, and hydrogen bonding.

Example 5

Scanning Electron Microscopy (SEM)

The surface morphology of unmodified and modified cellulosic materials was studied using SEM. The bacterial cellulose material was quenched in liquid nitrogen prior to freeze drying. The surface was then coated with gold prior to analysis, which was performed with a Zeiss DSM 940A operating at 10 kV.

Example 6

confocal laser microscopy

The morphology of bacterial cellulose in its wet state and the modification throughout the gel was studied using confocal microscopy equipped with a fiber-coupled ArKr laser. Filters are selected for the emission wavelength of the dye [λ _ex = 495 nm and λ _em = 516]. The wet bacterial cellulose sample was fluorescently labeled with fluorescently labeled xyloglucan (XG-FITC). XG-FITC was synthesized as described in Brumer, H. et al., Journal of the American Chemical Society, 2004. 126(18): pp. 5715-5721. The wet gel was stained with 2 mg/ml XG-FITC stock solution for 24 hours. After staining, excess XG-FITC was removed by placing the samples in deionized water overnight with gentle agitation.

Example 7

ESCA

ESCA was used to determine the chemical composition of bacterial cellulose before and after surface modification with xyloglucan. After modification and before measurement, the material was baked at 30°C. Measurements were performed using Quantum 2000 from Physical Electronics. The area to be analyzed is 500 x 500 μm² with a beam size of 100 μm. The angle between sample and detector is 45°. Peak intensities were measured and curve fitting was performed using MultiPak software from Physical Electronics. The characteristic ESCA spectrum of cellulose has one peak at 286.7 eV corresponding to a carbon single bonded to oxygen, and one peak at 287.9 eV corresponding to a carbon bonded to two oxygens. Relative amounts of different bonded carbons were calculated using Gaussian curve fitting of the highest resolution C1s peak. The different positions of C-C, C-O, O-C-O or C=O and O-C=O are 285.0±0.2eV, 286.7±0.2eV, 281.1±0.2eV and 289.4±0.2eV, respectively.

ESCA showed that the surface had been modified with xyloglucan. It can be seen that the amount of carbon bonded to the oxygen of the side group of xyloglucan slightly increases. However, the amount of GRGDS could not be quantified, probably due to the small size of this group and the possibility of embedding and pointing towards the interior of the gel upon drying. Trace nitrogen is also present in xyloglucan and cellulose itself, further complicating the characterization.

Example 8

Dynamic contact angle with water

Static contact angle measurements were performed on 6 dried cellulose films (unmodified and cellulose modified with XG or XG-GRGDS). A 5 microliter drop was applied to each cellulose surface. The contact angle θ _e is measured using a goniometer by recording the angle formed between the solid and the tangent to the drop surface.

Wettability was slightly higher when modified with xyloglucan and xyloglucan-GRGDS compared to unmodified BC (Table 1).

Table 1

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 relatively large contact angle with water of unmodified bacterial cellulose is due to the compacted structure, less pores (for capillary forces) and less available hydroxyl groups (due to high crystallinity). Modification with xyloglucan increases available hydroxyl groups and thereby improves wettability. The introduction of GRGDS does not significantly reduce the wettability, see the structure in Fig. 8. The wettability is still higher than that of unmodified BC.

Example 9

Protein adsorption using QCM

Sweden)研究蛋白质在纤维素表面上的吸附受表面改性的影响。Using QCM-D instrument (Q-sense AB,

Sweden) studied the effect of surface modification on the adsorption of proteins on cellulose surfaces.

Preparation of model cellulose surfaces on gold-coated QCM-D crystals. The surface was cleaned for 10 minutes in a UV/ozone chamber and then immersed in a 5: ₁ :1 mixture of Milli-Q water, _H2O2 (30%) and _NH3 (25%) at 70°C for 10 minutes. The surface was washed with Milli-Q and dried with nitrogen. Trimethylsilylcellulose (1 mg/ml in toluene) was spin-coated onto the gold surface at 4000 rpm for 1 min. The trimethylsilyl group was removed and cellulose was formed on hydrogen chloride vapor (10% solution). Measurements are made at the third harmonic (15Hz). The change in f reflects the mass coupled to the crystal surface. For thin, uniformly distributed rigid films, the adsorption-induced frequency shift (Δf) is related to mass uptake as described by the Sauerbrey equation:

$\frac{\mathrm{<span\; class="notranslate">\; m</span>}}{\mathrm{<span\; class="notranslate">\; A</span>}}<span\; class="notranslate">\; =</span>\frac{\mathrm{<span\; class="notranslate">\; Cf</span>}}{{\mathrm{<span\; class="notranslate">\; no</span>}}_{\mathrm{<span\; class="notranslate">\; r</span>}}}$

where m is the mass (ng), A is the area (cm ² ), n _r is the harmonic number (=1, 3...) and C is the mass sensitivity constant (17.7ng/cm ² /Hz). Measurements were performed in triplicate. Adsorb xyloglucan and xyloglucan-GRGDS at a concentration of 2 mg/ml. After the introduction of the cell culture medium, a step of desorption with water was performed after each adsorption. Protein adsorption was studied using the same medium as used in cell seeding. The cell culture medium contains a mixture of proteins, including the cell adhesion protein fibronectin containing the RGD motif. To elucidate whether modification with XG-GRGDS resulted in increased adsorption of proteins (particularly fibronectin) from cell culture media, adsorption studies were performed on model cellulose surfaces using QCM-D. A fibronectin antibody (Biotin (ab6584), Abcam) was introduced after introduction into the cell culture medium to confirm whether any possibly adsorbed protein was fibronectin. A desorption step with water is performed after all adsorption.

As shown in Figure 9, approximately 100 ng/cm2 of protein from the cell culture medium was adsorbed onto the unmodified cellulose surface. When xyloglucan was adsorbed first, no protein was adsorbed. When cellulose was modified with xyloglucan with an adhesive pentapeptide, less protein was adsorbed (50 ng/cm2) compared to unmodified cellulose. The introduction of fibronectin antibody IgG after modification with xyloglucan-GRGDS and cell culture medium. Anti-adhesin IgG did not adsorb, indicating that the adsorbed protein was not the adhesive protein fibronectin or at least not in the activated form.

Example 10

Wide Angle X-ray Scattering (WAXS)

的CuKα阳极。通过2θ＝5-30°进行扫描。测量非晶衍射的晶体衍射峰的强度。相对结晶度测定为晶体部分与总部分之间的比率。Freeze-dried pellets of BC were compressed into pellets 1 cm in diameter. X-ray diffraction patterns were recorded on a Siemens D5000 diffractometer. use wavelength

CuKα anode. Scanning was performed through 2Θ = 5-30°. Measure the intensity of the crystalline diffraction peaks that diffract from the amorphous. Relative crystallinity is measured as the ratio between the crystalline fraction and the total fraction.

Example 11

cell seeding

Sweden)，并将其保持在37℃下含5％CO₂的加湿培养器中。在细胞铺板之前，用相当于BC干重量的15％的木葡聚糖和XG-GRGDS将该BC改性过夜。在细胞铺板之前，将改性纤维素用PBS洗涤两遍。Endothelial cells (HSVEC) were isolated from healthy parts of the human saphenous vein using an enzymatic method. Cells were cultured in M199 (PAA Laboratories GmbH, Linz, Austria) supplemented with 20% fetal bovine serum (FBS; PAA Laboratories GmbH), penicillin-streptomycin (100U/mL; PAA Laboratories GmbH), 1.2mM L-glutamine (PAA), bovine brain extract (75mg/500mL; prepared in the laboratory), and heparin (13U/mL ;LeoPharma,

Sweden) and kept them at 37°C in a humidified incubator with 5% CO ₂ . The BCs were modified overnight with 15% xyloglucan and XG-GRGDS equivalent to BC dry weight before cell plating. The modified cellulose was washed twice with PBS prior to cell plating.

Germany)分析。用AxioCam HRc(Zeiss)对图片进行数字捕获。To assess cell morphology, HSVEC were plated on modified and unmodified BC plates at a density of 3 x ¹⁰⁵ cells/cm2. Samples were removed on Days 1 and 3 for evaluation. Cells were fixed in 3.7% formaldehyde and permeabilized in 0.2% Triton X-100. To make f-actin visible, use conjugated to Alexa Cells were stained with Phalloidin on 546 (MolecularProbes Inc., Eugene, OR, USA). Nuclei were counterstained with DAPI (Sigma-Aldrich Sweden AB, Stockholm, Sweden). The samples were mounted in Slowfade ^TM Antifade mounting medium (Molecular Probes Inc.) and Axio Imager M1 (Carl Zeiss,

Germany) analysis. Pictures were digitally captured with an AxioCam HRc (Zeiss).

cell adhesion

Initial cell adhesion studies showed that cell adhesion was faster and better on xyloglucan-GRGDS-modified cellulose than on unmodified and xyloglucan-modified cellulose. Optical microscopic images showed that more cells were present on the modified surface with more developed stretching and adhesion, see Figure 10.

Example 12

form

The morphology of cotton linters and bacterial cellulose differs in many ways. Cotton linters consist of fibers whose surface is covered with microfibrils. The fiber size is about 6 microns. Bacterial cellulose, on the other hand, is a swollen three-dimensional network composed of nanofibrils with a size of 70-100 nm.

Modification of bacterial cellulose with xyloglucan in the aqueous phase did not change this morphology (Fig. 11C). This is not the case if the modification is carried out in an organic solvent such as acetone, where the network shrinks significantly, compare Figures 11A and 11B. In order to maintain the network of BC, modification in water is preferred. Confocal Z-scan of bacterial cellulose modified with fluorescent xyloglucan (xyloglucan-FITC) showed that the nanocellulose material was uniformly modified throughout.

in conclusion

The inventors of the present invention describe a new method of modifying cellulose nanofibrils without affecting the morphology of the nanofibril network. Bacterial cellulose was successfully modified with xyloglucan-GRGDS as confirmed by colorimetry. The adsorption capacity reaches a maximum of 190 mg/g. The entire material of the nanocellulose material was uniformly modified as observed by SEM and z-scan in confocal microscopy. In addition, the modification in the aqueous phase is significantly more favorable for maintaining the morphology than in the organic solvent. This modification improves wettability, which may explain the reduced or negligible amount of adsorbed protein shown by QCM-D. Initial cellular studies have demonstrated improved adhesion of endothelial cells when BC hydrogels are modified with xyloglucan-GRGDS. As demonstrated by QCM-D, the improved cell adhesion was not due to the non-specific adsorption of fibronectin from the culture medium, but was due to the specific presentation of the RGD epitope by XG.

Although specific embodiments of the present invention have been described in detail by way of examples, it is apparent that those skilled in the art can make modifications and adjustments to the present invention. However, it is expressly understood that such modifications and adaptations are within the scope of the present invention as claimed.

Claims (20)

1. A method of preparing an implantable material by modifying a polymeric carbohydrate material by binding a carbohydrate linking molecule comprising a chemical group to the polymeric carbohydrate material, wherein the chemical group Imparting improved biocompatibility to said polymeric carbohydrate material, said method is characterized in that said polymeric carbohydrate material comprises cellulose, said method comprising the steps of: (a) providing a carbohydrate-linked molecule comprising a carbohydrate derived from xyloglucan and a chemical group that imparts improved biocompatibility; and (b) contacting said carbohydrate linking molecule with said polymeric carbohydrate material to be modified under conditions that bind said carbohydrate linking molecule to said polymeric carbohydrate material and improve its biocompatibility. 2. The method according to claim 1, wherein said carbohydrate linking molecule is provided by a method comprising the steps of: (a) providing xyloglucan-oligosaccharides; (b) covalently attaching a chemical group conferring improved biocompatibility to the reducing end of said xyloglucan-oligosaccharide; and (c) contacting the xyloglucan-oligosaccharide bearing the attached chemical group with a carbohydrate polymer derived from xyloglucan under conditions that result in the formation of a carbohydrate-linked molecule. 3. The method according to claim 2, wherein step (c) of forming said carbohydrate linking molecule is catalyzed by an enzyme having transglycosylation activity. 4. The method according to claim 3, wherein the enzyme is endo-xyloglucan glycosyltransferase. 5. The method according to any one of claims 2-4, wherein the xyloglucan-oligosaccharide contains 3-100 polymer backbone monosaccharide units. 6. The method according to claim 5, wherein the xyloglucan-oligosaccharide contains 4-10 polymer backbone monosaccharide units. 7. The method of claim 1, wherein the step of contacting and binding said carbohydrate linking molecule comprising a chemical group to said polymeric carbohydrate material is performed under aqueous conditions. 8. The method according to claim 1, wherein said cellulose is microbially derived cellulose. 9. The method according to claim 8, wherein the microbial cellulose is produced by Acetobacter xylinum. 10. The method according to claim 1, wherein said chemical group conferring improved biocompatibility is a protein or a peptide. 11. The method according to claim 1, wherein said chemical group that imparts improved biocompatibility to said polymeric carbohydrate material comprises at least one of the following: extracellular matrix adhesion molecules, growth factors, cell adhesion molecules, Anticoagulant factors or adhesion peptide fragments. 12. The method according to claim 10, wherein said peptide comprises an Arg-Gly-Asp containing peptide sequence. 13. The method according to claim 12, wherein the peptide sequence containing Arg-Gly-Asp is a peptide sequence of Gly-Arg-Gly-Asp-Ser; a peptide sequence containing Tyr-Ile-Gly-Ser-Arg; or Peptide sequence containing Ile-Lys-Val-Ala-Val. 14. An implantable material for medical or surgical use produced according to the method of any one of claims 1-13. 15. Use of the implantable material according to claim 14 for the manufacture of scaffolds for tissue engineering. 16. Use according to claim 15 for the manufacture of artificial blood vessels, artificial skin, nerve stents or orthopedic implants. 17. Use according to claim 15 for the manufacture of artificial blood vessels. 18. Scaffold for tissue engineering comprising a material according to claim 14. 19. An artificial blood vessel comprising a material according to claim 14. 20. The scaffold according to claim 18 or the artificial vessel according to claim 19, wherein said scaffold or artificial vessel has been pre-seeded with cells in vitro.

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