Hydroxyphenyl cross-linked macromolecular network and applications thereof

Mode of execution

In this terminology used in refers to the chain length poly-carboxylic acid ester containing at least two functional groups or units of the molecule, or like structure, in the element of the at least two such groups or units is or comprises carboxylic acid groups, in the in the pro-nuclear substitution reaction, the carboxylic acid group in the space can be close. In this same terminology used in refers to the chain length polyamine containing at least two functional groups or units of the molecule, or like structure, in the element of the at least two such groups or units is or comprises can be used in the nucleophilic substitution reaction is a primary amine. Similarly, in the poly-hydroxybenzotriazole molecular refers to the chain length for containing at least two functional groups or units of the molecule, in the element of the at least two such groups or units is or comprises hydroxy phenylic right, they can by C-C key and the other is connected with a hydroxy phenylic right. Similarly used in the preparation of the hydrogel is a network of material containing large molecule, macromolecular network is used for tissue replacement or engineering application, for example, as artificial cartilage, as surgical equipment in order to prevent tissue inflammation peridium material, or as a semi-permeable membrane such as artificial kidney.

The invention comprises a big molecular network a novel structure, the big molecular network through the adjacent long chain macromolecule hydroxy phenylic right is formed between, the connection formed between the large molecules effectively cross-linked so as to generate a huge network. Network basic cross-linking structure is shown as follows:

Wherein R₁ and R₂ are long chain macromolecule. R₁ and R₂ may be the same or different molecule, but will understand that in order to produce the appropriate network, in a network of the present invention at least a part of a dihydroxy benzene key R₁ and R₂ are different molecule. R₁ and R₂ not essential but is preferably the same kind of molecule.

Formed between the adjacent large molecules through a large amount of dihydroxy benzene key , generating the as shown in Figure 2 a dihydroxy shown in big molecular network divinylbenzene. In the diagram, with said cylindrical for macromolecules, each macromolecule comprising at least two of the hydroxy phenylic right connected with the. It is noted that each of the hydroxy phenylic right is not have to be connected with another hydroxy phenylic right.

Devices, the invention consists of compounds containing hydroxybenzene through carbodiimide-mediated through their reaction of a primary amine (or carboxyl) group is covalently linked to the various polymerization support material (or first-level amine) the carboxyl groups of the group, said compound including, but not limited to tyramine, the polymeric support material including, but not limited to, hyaluronic acid or chondroitin sulfate (for example, in the form of aggrecan). In the separation and purification of the hydroxy-substituted polymeric support, in the presence of dilute hydrogen peroxide, horseradish peroxidase (HRP) selectively undergo the hydroxy benzene remnant base cross-linked to produce hydrogel.

1st step preparation of macromolecular network is to prepare or provide the periodic zone hydroxyl phenylic right long chain macromolecule. In a specific embodiment, the macromolecules are provided with a plurality of or periodically with hydroxy phenylic right poly hydroxybenzotriazole molecule, such as polyphenol. Suitable polyphenol comprises a poly amino acid (such as poly tyrosine), table gallis catechol (EGC) and separated from the green tea in the table gallis catechol gallic acid (EGCG), other sub-optimal polyphenol.

In a further specific embodiment, the chemical reaction of hydroxy phenylic right can be periodically or randomly add on the long chain molecules. Hydroxy phenylic right will be added to the large molecule is one preferred method of using carbodiimide-mediated pathway in the substitution reaction with a hydroxy phenylic right level of amines and large molecule to form amide bond between the carboxylic acid group. In this method, a preferred long-chain macromolecule is periodic polycarboxylic acid ester of a carboxylic acid group of the molecule. Hydroxy phenylic right of the amine group is provided with a level a part of the small molecule, by carbodiimide means can be connected to the long-chain macromolecules in a carboxylic acid group on the carboxyl carbon atoms. Reactions are carried out are as follows:

Wherein:

Structure is a carbodiimide A;

B structure is poly carboxylic acid ester (although only marked a CO₂ H group);

C A the structure of the product of the reaction is, activated O-acyl isourea;

Structure containing hydroxy phenylic right D is a primary amine;

E structure is hydroxy substituted polycarboxylic acid ester;

Acyl urea by-product is F structure;

Wherein each R can be respectively selected, the same or different from each other, can be straight-chain or branched alkyl or acyl group, or any other structure, as long as these structure will not interfere with carbodiimide reaction pathway in NH₂ and CO₂ H E generated between the amide bond in the above-mentioned structure can be.

In the schematic of the above, the representative reaction A the carbodiimide activation of the carboxyl group so as to produce the activated O-acyl isourea intermediate. The intermediate chusei of the electrical behavior of the acceptable adjacent carbon atoms with hydroxy phenylic right amine member the level of the nitrogen atom of the nucleophilic attack on the electronic curvature. The nucleophilic substitution reactions (reaction B) of the product is a hydroxy substituted polycarboxylic acid ester and acyl urea by-product, acyl urea can thus get sufficient by dialysis to remove pure hydroxy substituted polycarboxylic acid ester product.

The above-mentioned carbodiimide reaction in the particular side reaction may occur, the ordinary technical personnel in this field should be made to this to be taken into account. First of all, carbodiimide can be with other nucleophiles reaction, rather than the generation of the necessary O-acyl isourea of poly-carboxylic acid ester molecules react with an oxygen atom of the carboxylic acid (reaction A). Other nucleophiles can include the above-mentioned structure in D phenylic right amine and/or hydroxyl group. In particular, reaction will occur A 3 so as to reduce the possible side reaction carbodiimide and with hydroxy a phenylic right effective concentration of level amine (structure and A D), and may result in the (B structure) gathers the carboxylic ether generating non-ideal adduct:

C reaction

Reaction D

Reaction E

The reaction product of a diimine amine and carbon (reaction C) no free amino, thus effectively reducing the can and O-acyl isourea tyramine of the amount of the reaction. The reaction also reduces the can produce the ideal O-acyl isourea of the amount of a carbodiimide. Hydroxybenzotriazole reaction product (reaction D) no UV absorption, this makes the final hydroxyphenyl-substituted polycarboxylate product (explanation see below) in the UV spectrum has become more difficult to observe. However, because these products still with a free amino group, they can be the reaction B and poly-carboxylic acid ester molecule produce the amide linkage. The reaction can produce two invalid hyaluric substituted structure, owing to the lack of generating free radical (explanation see below) can be required to extract the phenolic hydroxyl hydrogen atom, two structures are unable to participate in the preparation of the invention cross-linked network of cross-linking reaction in the 2nd step-peroxidase (description see below). The final, carbodiimide will be ineffective with water (reaction E) the response of the generated by-products with the above-mentioned reaction B same acylurea, however, there is no ideal product, the generation of E structure.

Once in the reaction generating A ideal O-acyl isourea product, and certain additional side reaction may occur:

Reaction F:

G reaction:

Reaction H:

O-acyl isourea (C structure) F shows reaction can be hydrolyzed, to release the original unmodified gathers the carboxylic ether (B structure) and carbodiimide the acyl urea (F structure). This reaction is similar to and the invalid reaction E, reduces the effective concentration of a carbodiimide. O-acyl isourea can also be through the intramolecular rearrangement (reaction G) generating two kinds of chemical inert N-acylurea. These structure of the molecule in the carboxylic acid ester adduct of generating invalid, the network cannot be used in the present invention in the preparation of catalytic cross-linking reaction of the peroxidase (steps discussed below 2). O-acyl isourea and can also be the same or different molecule poly-carboxylic acid ester of the carboxyl groups of the 2nd reaction (reaction H) generating anhydride. With the structure of the molecule can then be reacted to form ideal D amide and a 2nd re-obtain carboxyl groups. Therefore, for O-acyl isourea there are two possible side reaction, these side reaction carbodiimide can be reduced the effective concentration (reaction F and G) and poly-carboxylic acid ester molecule may result in non-ideal generated on the adduct.

The negative effect of these side reactions and non-due to an improper experimental method, but will take place through conventional operation.

In the above-mentioned means for the large molecule (B structure) gathers the carboxylic ether , in another alternative route, macromolecule can be is provided with a plurality of or periodic with amino group of the polyamine, as a carboxylic acid wherein a hydroxyl group phenylic right part of a small molecule. Suitable polyamines include: poly hexosamine such as chitosan (poly glucossamine); poly-amino acid such as polylysine; poly DNA such as polyethylene (dA) (poly deoxyadenilic acid), poly (dC) (poly deoxycytidylate), poly (dG) (poly deoxy guanylic acid); and poly-ribonucleic acid such as (A) (poly adenylate), poly (C) (poly butcher glucoside acid) and poly (G) (poly-guanylate). Carbodiimide-mediated responses completely in accordance with the explained above for the reaction route, thus the amino group and carboxylic acid groups to form amide bond between, the only difference is the generated product is hydroxy substituted polyamine but not poly-carboxylic acid ester, this is the ordinary skill in the art will understand. Other peptides and/or protein can also be used as large molecules of the invention, as long as its with hydroxy phenylic right, or by the substitution reaction of the described here can be obtained can be hydroxy substituted group. For example, peptides of the invention have been described in addition, poly-arginine can be used as a large molecule.

When the carboxylic acid ester is also substituted on molecules, suitable for use in this invention containing hydroxy compound includes a compound with a free first-level amine , can be used for modified first-level amine with multiple or periodic with CO₂ H perssad the support material, including tyrosine (2-amino-3-(4-hydroxyphenyl) propionic acid) and tyramine (tyramine or 2-(4-hydroxyphenyl) ethylamine). When the polyamine is occurring, appropriate hydroxy-containing compound include with free CO₂ H group compound, CO₂ H group for modification can be used with multiple or periodic with a level NH₂ perssad the support material, including tyrosine, 3-(4-hydroxyphenyl) propionic acid and 4-hydroxy-acetic acid.

According to the invention for preparing cross-linked macromolecular network of the 2nd step is the dihydroxy connecting structure has been obtained with one or more of hydroxy phenylic right is connected with the large molecule. In this step, different macromolecule hydroxy phenylic right through the following reaction mechanism in the presence of peroxidase, connect using the oxide reagent:

(It should be noted that some dihydroxy-benzene can also be connected in the same molecule is formed between the different hydroxy phenylic right. ) In dilute peroxide (preferably H₂ O₂) peroxidase can be from the presence of the compound with a hydroxy (such as tyramine) extracting hydrogen atom of the phenolic hydroxyl group, phenolic hydroxyl group to the oxygen atom one fatherless electronic, become extremely active radical. The free radical the two equivalent ortho-carbon atoms an isomerization, then two such dimerization taken place in the structure of forming the covalent bond will structure can effectively cross-linking, cross-linking structure to produce the enol dimer dihydroxy-benzene (following note dihydroxybenzonic benzene key such as di tyramine linkage).

The interest of brevity, a single display only more than two hydroxybenzotriazole is connected with the reaction, however, it should be understand that when the with the hydroxy phenylic right large molecules are placed under the reaction conditions (superoxyde and peroxidase) will be generating a plurality of or multiple such a key. The hydrogen peroxide used in the above-mentioned mechanism, but may also use other suitable peroxide. Similarly, peroxidase preferably horseradish peroxidase (HRP). Alternatively, the free radical can be generated with hydroxy phenylic right of undergoing a cross-linking of the support material of any other suitable enzyme (or other reagent) can be used, in the following ordinary metabolic condition in order to optimize the function.

The crosslinking reaction is the enzyme actuation (peroxidase), dihydroxy divinylbenzene the big molecular network is superior to the traditional cartilage or other tissue replacement or substituted method and product. enzyme actuation means that the cross-linking reaction in the ordinary vivo or metabolic conditions, temperature is 35 the [...] -39 the [...] (for example, the 37 [...]), in the range of pH 6-7 (for example, 6.5), a reaction reagent, and so on. (Peroxide, such as hydrogen peroxide, is cross-linked in the reaction reagent required reaction only). Therefore, the crosslinking reaction can be carried out in vivo, in the surgical site such as orthopedic surgical site offers cross-linked hydrogel, thus contributing to hydrogel and natural tissue such as bone or cartilage tissue between the dense integration to the greatest extent. Since the hydroxy substituted large molecule cross-linked before the rapidly permeate into the already existing cartilage matrix, not only with other hydroxy substituted macromolecular support material undergoing a cross-linking, have been and may also be present in the cartilage matrix protein tyrosine residues in a cross-linked, so the new hydrogel bracket and the integration of the natural cartilage matrix can rapidly occur. In this way, can eliminate the use of pre-made substrate bolt; the typical problem found, that is, they are difficult to integrate into the natural cartilage tissue. Directly cross-linked hydrogel can be in the joint surface is not longer needed, the operation wound expanded to adapt to the bolt is, for those chemical reaction or otherwise toxic to the patient in the patient's body to form the hydrogel must be obtained in advance tumbler. It should be noted that most of the arthritis has caused cartilage damage is a joint surface of the thinning of the different degrees and is not a hole with a certain shape.

The crosslinking reaction needs to be peroxide and peroxidase (preferably horseradish peroxidase), in order to conveniently applied to surgical site, can be prepared containing all component solution only lacks a component. For example, can be prepared containing tyramine (or other types of containing hydroxybenzene) substituted gathers the carboxylic ether (such as tyramine-substituted hyaluronan, etc.) solution and peroxidase, and preparing the 2nd solution containing peroxide. Optionally, in the 1st and 2nd solution of peroxide and peroxidase can be exchanged, it is important that prior to the occurrence of the crosslinking reaction of peroxide and peroxidase preserved separately (that is, in a separate solution). Subsequently, using 1st solution, (for example, in in vivo surgical site), and the 2nd solution using in vivo on or spray on the 1st solution, the tyramine residue home position spot formed of cross-linked. Cross-linking reaction occurs in vivo. Recorded according to the present invention, the other for the ordinary technical personnel in this field will be obvious.

Furthermore, because the cross-linking reaction under the ordinary metabolic conditions, the additional of live cells can be, such as the cartilage cells, progenitor cells, stem cells, containing uncross-linked directly to the hydroxy substituted polycarboxylic acid ester or polyamine (or polyphenol) in a culture medium, that is, in the front section of the 1st or 2nd solution, wherein the rich in cell culture medium to be used in conjunction and site in vivo, molecules in the subsequently occurring after adding peroxidase and peroxide cross-linked. The big molecular network after cross-linking of the dispersed in an ideal cell. Traditional substrate because of preparing the conditions of extreme temperature and pH, rich in cell network of this kind is not possible in the conventional tissue replacement matrix. Furthermore, as the following embodiment 5 described in, the above-mentioned invention has been confirmed in the substrate according to the present invention in the cells of a tyramine-substituted hyaluronan cross-linked (description see below) after generating network still alive.

In is particularly fit for the preparation of synthetic cartilage and other synthetic or artificial tissue of the preferred embodiment, according to this invention used for generating network of the macromolecule is hyaluronic acid (HA), and hydroxy phenylic right provided in the form of to tyramine.

As shown in Figure 3, HA β-1 by repeated to, 3 glycosidic bond the glucuro (glcA) and is connected with-acetyl glucosamine N the residue (glcNAc). Hyaluronic acid chain for each section, the simple disaccharide repeat up to 10,000 times, between each repeating disaccharides through β-1, 4 glycosidic bond connection. Each of the glucose ring in its glcA 5 carbon atoms with a carboxylic acid group is (CO₂ H). Tyramine is OH group with the benzene ring is a phenolic molecule ethylamine group. When the use of these elements, the tyramine substituted to HA the CO₂ H group on the oxygen atom of the mechanism shown, is through the above-mentioned carbodiimide-mediated reaction mechanisms. The preferred carbodiimide class is indicated by the 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC).

Wherein:

Structure is A EDC;

Structure B is hyaluronic acid (although only one showed CO₂ H);

C A structure is a product of the reaction of 1-ethyl-3-(3-dimethylamino propyl) isourea;

D structure is tyramine;

E structure is tyramine-substituted hyaluronan;

Structure is F 1-ethyl-3-(3-dimethylamino propyl) urea (EDU).

In the above-mentioned route, carboxyl groups of the hyaluronic acid molecule the electronegative oxygen atom through nucleophilic reaction mechanism (EDC) carbodiimide molecule attack on the carbon atom diimide electronic of generating activated O-acyl isourea (reaction A). Therefore HA the carbon atoms in the carboxylic acid ester group to the electronic deficienty make it vulnerable to tyramine molecule amine perssad lone electronic attack (reaction B). Preferably, reaction A by appropriate catalyst activation is generated in the reaction of ester A, the reaction can be in a substantially neutral pH (such as pH=6.5) to under. Appropriate catalyst includes N-hydroxy succinimide (NHS), sub-optimal 1-hydroxy benzotriazole (HOBt) or N-hydroxy sulfo-succinimide (NHSS), sub-optimal other by forming activates ester effectively improve the carbodiimide reaction makes the carbodiimide in the higher the pH the invalid hydrolysis minimize the appropriate catalyst or mixture of them. The EDC can be used in addition to other good carbodiimide including 1-cyclohexyl-3-[ 2-(4-methyl-morpholine) ethyl] carbodiimide (CMC) and dicyclohexyl carbodiimide (DCC).

The above-mentioned reaction A generalum the O-acyl isourea-substituted hyaluronan; EDC molecule Provisional substituted to HA glcA residue in the molecule is a carboxylic acid group, the carbon atom of the carboxylic acid group with a weak positive electrically. As explained in the section, electronic amine group of tyramine molecule through the subsequent nucleophilic substitution reaction to the carbon atom of the replace (reaction B). The tyramine substitution reaction generating B HA molecular (T-HA) and spinoffs acyl isourea. Should be aware that the response in the B HA A and periodic glcA residue of generating a plurality of tyramine substituted; for simplicity considerations in this only display a single substituted.

If the above description and explanation, in the generation after T-HA, a plurality of molecular T-HA phenolic through superoxyde and peroxidase molecule T-HA in addition. Molecular HA of the hydroxy group of tyramine residues is connected with the presence of peroxidase phenylic right and peroxide (preferably H₂ O₂) phenol thereby removing reaction generating tyramine free radical hydroxyl hydrogen atoms, the oxygen atom of a on phenol matching electronic. The free radical generating isomerization or resonance, the resonance structure is generated (or free radical isomer) in the matching electron transfer to phenol ring ortho-carbon atom. In this position, not matching electronic rapidly and another tyramine free radical on the similar position not matching electronic generating a covalent bond. Its result is at the same or different HA molecules on different glcA residue between the different tyramine free radical to generate a free radical driving two polychem. This dimer to produce the enol the tyramine residues are connected with each other, generating di tyramine linkage structure. Should be made clear in the adjacent tyramine residue is joined will be a lot of the reaction, molecular T-HA of the invention is the cross-linked macromolecular network, the network has the following cross-linking structure:

Cross-linked T-HA the network can be connected through conventional method such as by adding protein aggrecan, generated in the network of cross-linked T-HA the aggrecan molecule is connected with chain HA. Therefore, according to the present invention can be obtained, and the normal cartilage aggregate similar to the network in the network, wherein the di tyramine linkage of cartilage replace normal collagen fibers in the network are connected together and containing by this restriction of the aggrecan network.

By this invention, other mucopolysaccharide, polysaccharide and poly-carboxylic acid can also be used as a large molecular generating in this record of the cross-linked network. For example, in addition to the appropriate mucopolysaeccharide outside HA including chondroitin, chondroitin sulfate, dermatan sulfate, heparan sulfate and heparin. Other suitable polycarboxylates include: proteoglycan such as multifunctional protein chitosan, aggrecan and by the aggrecan, hyaluronic acid and connecting up of the protein to cartilage aggregate; chitosan halfaldehyde such as pectin acid salt (polygalacturonic acid), poly glucuro, pectin (polygalacturonic acid methyl ester), poly-sialic acid (poly [2, 8-(N-acetyl neuraminic acid)]) and alginate (mannuronic acid and ketogulonic halfaldehyde copolymer); and the definition of the above-mentioned carboxylic acid ester with the amino acid (with at least 2 amino acid unit), such as polyaspartic acid, polyglutamic acid. In this record of the reaction channels bonded by urea groups-mediated, the ordinary technical personnel in this field through the correct experimental method for all these large molecules to carry out one or more of hydroxy substituted phenylic right.

As mentioned above, it should also be understood that the natural polyphenol compounds already containing two or more hydroxy groups phenylic right, hydroxy phenylic right through the enzyme may be cross-linked catalyzed chemical reaction, therefore, to substitute the above-mentioned polyphenol compounds can be by chemical reaction with the hydroxy phenylic right and polyamine polycarboxylic acid ester.

In another preferred specific embodiment, using tyramine cross-linked chondroitin sulfate molecules (independently or as aggrecan a portion of the) network is used for simulation or replacement of normal cartilage. Chondroitinsulfate and hyaluronic acid the same, in addition to:1) repeated disaccharide structure comprises is N-acetylgalactosamine (galNAc) instead of glcNAc, the only difference lies in the 4 carbon hydroxyl group (in fig. 3 marked with a circle); 2) the O-sulfate residues galNAc 4 and/or 6 and/or of glcA residue 2 hydroxy (Figure 3) on and 3) chain length of chondroitin sulfate, hyaluronic acid less than the 20 [...] 100 repeated disaccharide units. (Aggrecan molecule is composed of a plurality of-about 100 of sulfuric acid chondroitin chain through each chain of reduction at the end of the is connected with the core protein connection sugar). In the specific embodiment, the adjacent (cross-linked) chondroitinsulfate molecular electronegative SO₄^2- group the network that the repulsive force produced agglomerates having pressure resistance and tyramine cross-linked to prevent chondroitinsulfate network broken or spallation. Thereby generating the normal cartilage in the similar irreplaceable chondroitinsulfate network (accompanied by and impermeable), however, there is no normal cartilage in the original other glue the ectodomain of the fiber matrix or HA chain. In fact, by directly cross-linking chondroitin sulfate molecules (rather than the aforesaid specific embodiment the core of the molecule HA), adjacent chondroitin sulfate enhanced repulsion force between the molecules, so that the convection flow less than the normal cartilage stronger resistance. In the flow of the fluid due to the clearance more restricted, directly cross-linked chondroitin sulfate molecules to absorb and disperse the capacity of the loading force is more than that of the normal cartilage is stronger. The chondroitin sulfate molecules are directly cross-linked the specific embodiment, some cartilage degradation is the condition of complete control; for example, under certain conditions the normal cartilage with chondroitin sulfate molecules in the core protein in normal connected HA binding domain (G1) and the 2nd spherical domain (G2) fracture occurs between, then chondroitin region diffuses from the cartilage in the aggregate. In the specific embodiment, the chondroitin sulfate without directly cross-linked between molecules with aggrecan or other protein is connected with the chitosan molecules, as they will not be in the normal cartilage is cut off or removed.

However, due to the high degree of HA can be utilized, preferably tyramine cross-linked T-HA network (with aggrecan molecule HA chain, aggrecan molecules include chondroitin sulfate chain). This when the invention is in cartilage replacement or repair may be useful, because physical generating gristly normal metabolic pathway can be used directly on the implanted tyramine cross-linked T-HA formed on the network, the following will explain.

The above-mentioned two tyramine cross-linked T-HA network for the production of artificial or synthetic cartilage has special use. Cartilage transplant often used for head and neck reconstruction process in order to repair the trauma or congenital malformation of the cartilage or bone defects in. The specialized for ear auricle and ear application of the reconstruction, repair is frequently used in this application due to trauma, tumor (in other words, squamous cell carcinoma, basalioma and melanoma) and inherent defects such as small ear sickness formed. Specialized for nasal application including nose and nasal septum modification and reconstruction process. In the commonly used and increased nose, nose cartilage (tipgraft), cover cartilige (  graft shield) and supporting cartilage (  graft spreader). The trauma, tumor, auto-immune diseases such as Wegeners granulomas or congenital defect of the cartilage repair nasal reconstruction needs. Diaphragm perforation difficult to treat and regular treatment failure. The cartilage grafting used for these treatment is very ideal, but autologuous or donated cartilage often shortages. Specialized in view of throat applications include laryngotracheal reconstruction, cartilage takes the rib for children usually required, this is also dangerous. Ear and diaphragm cartilige for the purposes of this kind of application is often not enough. In the description of the network formation of cross-linked HA engineering cartilage, in addressing these issues have a major impact. throat trachea reconstruction is frequently used in the treatment of the subglottal or pipe caused by the narrow air passage narrowing. The disease can be traumatic (i.e. cannula damage or decannulated) or congenital.

Other application also includes the chin and the cheeks is increased, and lower eyelid evaginable repair a large number of craniofacial application. It should be noted that in these applications the cartilage of the articular cartilage and it may not be necessary to have a completely identical mechanical properties. At the same time may wish to which may include cells or biological active reagent.

The cross-linked network of the present invention play a major role in a special application is the production of artificial kidney. Kidneys filtering the blood through two mechanisms: one through size exclusion, charge exclusion of the other. The MEMS device has been designed for use in artificial kidney device, device contains a precise definition of microporous, microporous only can effectively simulate the size of the kidney is ruled out. In a healthy kidney, on the charge of the filtering is to exist in the basement membrane heparan sulfate proteoglycan, the filter the two different types of cell separation, other related to the kidney function is very important. MEMS engineering of artificial kidney in order to simulation this kind of charge barrier, can be prepared containing sulfuric acid acetyl heparin or heparin hydrogel, the hydrogel through the dihydroxy described herein the power on (two tyramine) cross-linked, and into the hole of the MEMS device. The heparin/heparan sulfate hydrogel can then be clamped in the two layers of the hyaluronic acid hydrogel (such as the above-mentioned the T-HA) in, two layers of hydrogel containing kidney usually exists in the normal function of the kind of cell. In the middle of the heparin/heparan sulfate hydrogel as the device provides the capacity of the charge exclusion. The outside two layers of hyaluronic acid hydrogel protection of the intermediate layer from the immune system violations and normal cells and the pollution of the molecular fragments. Two sides of the filtration barrier of the two types of cells to normal physiological direction provides the cell component.

In another promising application, the hydrogel of this invention can be used for the development of artificial pancreas. The development of artificial pancre one problem is due to the dirt of the in vivo monitor electrode, the half-life of the MEMS engineering glucose sensor is very short. In these monitor in this covered on the upper surface of the hyaluronic acid hydrogel (in other words T-HA) will allow detection of the proliferation of small molecular weight glucose molecules, at the same time, however, can protect the sensor from the immune system violations and normal cells and the pollution of the molecular fragments.

In short, according to the aforesaid it is evident that as the support material of a hydrogel is formed large molecule including, but not limited to, poly-carboxylic acid ester (containing free carboxyl groups), polyamine (amine containing free level), polyphenol (containing free hydroxyl phenylic right) and their interpolymer, for example already described above. When using many when phenol , the above-mentioned preparation of the invention can omit the 1st step of the network, because polyphenols already includes a plurality of or periodic hydroxy phenylic right. On the contrary, must be and polyamine polycarboxylic acid ester preferably through the above-mentioned carbodiimide reaction way to add or replace the hydroxy phenylic right on. In order to form a cross-linked structure, 2nd step is preparation of the network in the adjacent large molecule (whether gathers the carboxylic ether , polyamine or polyphenol) of between two hydroxy phenylic right enzyme actuation dimerization reaction. The steps in the metabolic temperature and under the conditions of pH, in the appropriate (preferably HRP) enzyme presence of peroxide reagent of (preferably hydrogen peroxide).

In a preferred two tyramine cross-linked in the network T-HA, in the 1st step (HA) high molecular weight hyaluronic acid in the tyramine substituted by carboxyl groups, tyramine the active hydroxy phenylic right HA molecule is introduced. The tyramine substitution reaction is preferably made of carbodiimide, 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC) mediated, HA on the degree of tyramine substitution of tyramine from the reaction mixture, the molar ratio of EDC and HA decision and absolute concentration. Excessive reaction reagent if no reaction occurs then the tyramine and EDC by dialysis to remove, thus separation and recovery of tyramine substituted HA(T-HA) of the high-molecular weight. In the preparation of the various T-HA tyramine substituted percentage can be calculated through the following simple measurement to:1) the concentration of tyramine in the preparation, according to tyramine at 275 nm UV absorption property can be a specific spectroscopic quantitative (see embodiment 2); and 2) in the preparation of HA the concentration of total carboxyl groups, by the standard of a hexose halfaldehyde analysis spectroscopic quantitative. Through this technology, only contains 4-6% tyramine substituted of already T-HA through the experiment of the conventional synthetic. In this tyramine substituted level, most of the molecules in HA (preferably at least 60, 70, 80, 90 or 95%)chemically unchanged, thereby still maintaining biological function. Based on the prescription T-HA (in other words 4-6% tyramine substituted), by simply changing process of the 2nd step T-HA in the concentration can be used with a wide range of physical properties of a wide range of biological material.

In the cross-linking reaction, enzymatic solution T-HA driving reaction (peroxidase) cross-linked to form the hydrogel, the molecule HA catalyzing the reaction of the adjacent two tyramine adducts covalent bond is formed between the, generating single two tyramine cross-linked. In the molecule each HA by generating hundreds of such two tyramine cross-linked so as to generate a stable three-dimensional support or hydrogel. The initial need to add cross-linking reaction is very dilute peroxide (preferably H₂ O₂), true because peroxidase substrate is a peroxide, rather than HA. Peroxidase to the oxide reaction product is composed of a tyramine hydroxy benzene ring priority obtaining free radical, thereby generating two tyramine cross-linked. Two tyramine the connecting structure has a blue fluorescence (see embodiment 2), the properties can be used in hydrogel imaging and measurement of cross-linking degree of the hydrogel. The crosslinking reaction is the enzyme actuation , the hydrogel can be formed under physiological conditions, can be included in the formed active preparation of cells or organisms, or directly with the living body tissue adjacent and keep the activity of the cells and tissues.

Generation of visual is transparent on the hydrogel, according to the concentration of initial T-HA different can have various physical properties. For example, experiments show that by containing 6.25, 12.5, 25, 50 and 100 mg/ml of   T-HA T-HA solution to form the hydrogel are respectively provided with a jelly, gel, dough, such elastic rubber composition (similar to the rubber balls) and types of cartilage material-see the physical properties of the embodiment 3. These materials under a wide range of clinical conditions has application potential, including for orthopedic (in other words, cartilage, bone, tendon, meniscofemoral, such as intervertebral disc) and non-reshaping surgical (kidney, liver, pancreas, etc.) organized tissue engineering, gene and drug delivery, non-biological device of the in the body (i.e. glucose sensor, such as artificial heart) the coating, wound repair, reconstruction and biological inductor design.

Said hydrogel the excellent properties include: 1) can simply identifying and quality control; 2) can be integrated with existing tissue matrix; 3) can be directly integrated into the new generation matrix; 4) can directly comprising cells and biological active factor; 5) can maintain biocompatibility; 6) is capable of controlling the biological reabsorption; 7) can form a complex in the anatomic of glue (see the following embodiments 6); and 8) is capable of assuming natural organizations, such as the mechanical properties of articular cartilage.

Combining one or more of the following embodiments of the present invention have a better understanding of various aspects of the, in this embodiment as an example.

Embodiment

Embodiment 1

Experimental amount of this invention has two tyramine cross-linked tyramine-substituted hyaluronan hydrogel prepared according to the following method. Based on hexose halfaldehyde of HA 1 mg/ml concentration dissolvedin 250mM2-(N-morpholine) ethyl sulfonic acid (MES), 150 mm   NaCl, 75 mm   NaOH, pH6.5 in the solution, containing in solution relative to the molar concentration of the carboxyl group HA 10 times excess of tyramine. HA carboxyl group subsequently joined relative to the molar concentration 10 times the initial EDC excess of tyramine substituted on the carboxyl group. The molar ratio of the molar amount of EDC 1/10 the N-hydroxy succinimide (NHS) into the reaction through a catalytic EDC activates esters to assist in the conduct of the amidation reaction. Reaction at room temperature for 24 hours, then through successively to 150 mm   NaCl and UPW thorough dialysis and freeze-drying has occurred the reaction of molecules from the small molecular weight reaction reagent such as tyramine, EDC, recovering and NHS MES. After the freeze-drying, according to the required hardness of the final hydrogel, the tyramine substitution product to HA(T-HA) the 5 [...] 100 mg/ml concentration dissolvedin PBS the operation between the (the buffer solution with the cell suspension, body tissue contact and compatible with the cross-linking reaction) is carried out in preparation of different concentrations. Can be selected, the solvent may be in addition to any other than the PBS of appropriate solvent, as long as the solvent has no negative influence on the basic enzymatic activity and will not be through the selective absorption enzymatically generated free radical cross-linking reaction, the occurrence of interference. Suitable replacement solvent including water, traditional biological tissue culture medium and cells refrigerating fluid (usually by about 90% serum and about 10% of the dimethyl asia animato sound of wind). The cell suspension (see example 5) or before contact with the tissue in vivo, by T-HA should be 0.2 the filter   m filter. In each subsequently added in preparing T-HA 10 U/mL type   II horseradish peroxidase (HRP) is connected with to tyramine-tyramine. By adding small volume (the 1-5  l) dilute hydrogen peroxide solution (0.012%-0.00012% final concentration) initial cross-linking in order to produce the desired hardness of the final hydrogel. In order to prepare a greater amount or large-volume ideal hydrogel, the ordinary technical personnel in this field can be provided in this section of the appropriate amplification reagent.

Embodiment 2

Experiments confirm this invention the T-HA macromolecular network of tyramine substituted (and subsequent two tyramine cross-linked) degree. First, preparation according to the above-mentioned method 3 formulation (uncross-linked) tyramine-substituted hyaluronan (T-HA), named 0X, 1X or 10X. 0X EDC preparation of the formulation is not used (i.e. not containing carbodiimide), implies that there is no carbodiimide-mediated tyramine NH₂ group and HA molecular CO₂ H group amide linkage is generated between the reaction. Therefore, 0X formulation can be considered as the control. 1X formulation in the reaction mixture containing the molecule HA CO₂ H groups and bases quantity stoichiometric ratio of 1:1 of the EDC. 10X formulation in the reaction mixture containing the molecule HA CO₂ H groups and bases quantity stoichiometric ratio of 10:1 (or 10 times excess) of EDC. In all three formulations, tyramine of relative to stoichiometric excess is of HA CO₂ H the number of the purposes of groups. In all three formulations (0X, 1X and 10X) in, a reaction reagent and formulation an appropriate amount of EDC in mixed in the reagent bottle and wabbling accelerate the tyramine substitution reaction. All of the formulation of the reaction at room temperature 24 hours, followed by dialysis to remove the contents of the reagent bottle in the tyramine molecule not react, acyl urea (EDU) EDC and reaction by-product. The tyramine, EDC and EDU HA the volume of the case with respect to the large molecules is relatively small, by dialysis with these elements it is very apt to have been generated and any HA T-HA molecular separation. Once the reaction had not been removed the tyramine and EDC, a residue of the to each formulation for HA analyzed to determine of all of the available CO₂ H alantin tyramine of the total number.

Tyramine at 275 nm UV absorption peak appears, according to tyramine standard curve can easily observe the tyramine substitution degree. According to the above-mentioned three kinds of T-HA of the formulation in the absence of UV spectral analysis found in the presence of EDC (formulation 0X) of the HA-tyramine substitution reaction of the molecule HA tyramine substituted is almost 0. The results confirmed in the tyramine substitution reaction using carbon in the importance of the carbodiimide reaction means. However, in the tyramine substitution reaction 1 : 1EDC: CO₂ H the stoichiometric ratio in the preparation of formulation T-HA tyramine absorption display HA on the chain of all available CO₂ H group of tyramine alantin is about 1.7%. 10X formula (10 : 1EDC: CO₂ H) caused approximately 4.7% alantin of.

Subsequently, the hydrogen peroxide and horseradish peroxidase (HRP) to 5 mg/ml is added to the concentration of the three dialysis in the formulation HA/T-HA (0X, 1X and 10X), generating the formulation of the reaction occurs until the reaction is complete. The addition of a peroxide and HRP observed after the completion of the reaction of 0X formulation still maintain full liquid, has obvious meniskus; the formation of gel have not been observed, confirmed in the absence of the use of EDC in the tyramine substitution reaction is free or substantially no tyramine substituted facts. 1X formulation only to weak meniscus surface content of the reagent bottle has been gelled, the confirmed tyramine substituted and cross-linked. 10X formulation forms a hard gel, in fact relative to the container of the liquid volume, in a certain amount of the liquid is arranged at the upper part of the same (with the meniscus). 10X of the gum preparation (with 4.7% of tyramine alantin) than with 1.7% tyramine alantin the 1X formulation hard-many.

Two tyramine structure presenting exposed to the UV light of the blue fluorescence. The product of the above-mentioned each formulation is exposed to the UV light in order to observe the existence of two tyramine cross-linked. And it is expected that the same, 1X and 10X hydogels the blue fluorescence (10X hydrogel fluorescence ratio 1X more strong fluorescence of the hydrogel), but 0X formulation no blue fluorescence. Results confirmed the 1X and 10X there are two tyramine cross-linked hydrogel, the hydrogel of the relatively hard (10X) than that generated in the tyramine of soft hydrogel (1X) more.

The overall result proves that the bonded by urea groups-mediated the importance of the reaction channels, in the invention is confirmed by cross-linking the hydrogel network forming T-HA the relative hardness and two tyramine cross-linked is proportional to the degree of, two tyramine cross-linking degree of tyramine substitution with HA is proportional to degree. A surprising and unexpected result is that according to the present invention, even if 1.7% of tyramine alantin (and subsequently formed connection rate two tyramine of) the appropriate intensity can still provide T-HA gel (or hydrogel). 4.7% alantin of (and cross-linked) or even generating a more firm T-HA gel. Equally surprising is that of the reaction mixture relative to the carboxylic acid in the presence of stoichiometric groups and bases quantity 10 times excess of the uses (formulation 10X) imide (EDC) caused by only about 4.5 the [...] 4.7% of tyramine alantin, although can obtain a stable and of tyramine cross-linked T-HA network.

These results mean that although the generated network is a stable hydrogel, the molecule HA most of the carboxylic acid group not substituted and tyramine cross-linked but basic natural HA and the like in the molecule. Therefore, when the network of the invention the substituent as cartilage in vivo, as compared with in the normal cartilage HA, network or T-HA of this invention most of the molecules in the gel have not basically changed HA, people believe that body's natural metabolic pathway (needing or not needing T-HA in the network with the help of the cells) of the network of the present invention can be considered as natural biological material and to its associated common synthesis and metabolic function. Furthermore, it was noted that in the body generally is a highly HA of the material and a material is not generated in the human body. Therefore, people believe that contain a large number of the present invention does not change the natural HA the cross-linked macromolecular network of in a wide range of the desired or to provide combined in the human body tissue engineering of the organization has important application in the application. This marks the remarkable progress in the technical state. Therefore, very let people amazedly, a high degree of tyramine substitution, for example, greater than about 10-20% substituted may not be desired; the above-mentioned experiment proves that generates the proper T-HA network does not require a high degree of substitution. Preferably, dihydroxy-benzene of the present invention (such as two tyramine) cross-linked poly-carboxylic acid ester (for example HA) network based on poly-carboxylic acid ester of the molecule (HA) CO₂ H hydroxy (tyramine) of the total amount of the percentage of alantin of less than 50, preferably less than 40, preferably less than 30, preferably less than 20, preferably less than 15, preferably less than 10, preferably less than 9, preferably less than 8, preferably less than 7, preferably less than 6, preferably less than 5.

Embodiment 3

Traditionally, it has been believed that natural cartilage has thermoviscoelastic, and mainly aggrecan matrix in the presence of chondroitin sulfate chain of electronegativity adjacent SO₄^2- the repulsive force of the between groups, natural cartilage is able to resist deformation and to absorb compression load. Tests confirm this invention only contains two tyramine cross-linked hyaluronic acid molecule (i.e. with no aggrecan and chondroitinsulfate) in the absence of large molecular network SO₄^2- group compared with the natural cartilage in against deformation and absorb the effects of pressure. According to embodiment 1 for the preparation and purification of non-cross-linked with about 5% tyramine alantin of formula T-HA. Based on the prescription T-HA, preparing five different T-HA concentration:

Concentration 1 : 6.25 mg   T-HA/ml   PBS;

Concentration 2 : 12.5 mg   T-HA/ml   PBS;

Concentration 3:25 mg   T-HA/ml   PBS;

Concentration 4:50 mg   T-HA/ml   PBS;

Concentration 5:100 mg   T-HA/ml   PBS.

The preparation then the above hydrogen peroxide and horseradish peroxidase reaction in the presence of, and embodiment 1 in the same molecular T-HA formed between the corresponding two tyramine cross-linked so as to generate a hydrogel 1, 2, 3, 4 and 5. Surprisingly and unexpectedly discovered that in the preparation of the concentration of T-HA according to different, five hydrogel become stable with different physical properties and basic adhesive material. For example, concentration 1 generated hydrogel has and vaseline or jelly considerable hardness and rheological properties; hydrogel stability and adhesion, however, a such as a spatula or other traditional tools to apply the external force or spread can be caused to flow. Hydrogel 1 has excellent viscous, is the surgical such as eye surgery surgical department the non-allergic peridium material ideal candidate material. Hydrogel 2 than hydrogel 1 more hard, this is because in the preparation of higher concentration of T-HA foreseeable T-HA and subsequently the increase of the density of the cross-linked in the molecule caused by the reduction and the increase of the cross-linked between molecules. Hydrogel 2 shows gel characteristic rehologic and the hardness of the nature, for the external load and to a certain extent elastic sticks instead. Under a greater load, hydrogel 2 will not flow but breaks into small debris, this is also the characteristic of the gel material. Hydrogel 3 has a dough or can extend the nature and consistency of the dough, when the applied external force not flow. And the hydrogel 1 and 2 compared with, this material demonstrates the stronger thermoviscoelastic. Hydrogel 4 are highly hard and adhesive gel, can be very strongly would not rupture resisting the external force. Hydrogel 4 is highly elastic rubber-like composition, the sudden pressure (such as by falling on the floor) can be generated under elastic. Hydrogel 4 this kind of sudden pressure to the capacity to produce elasticity in some joint replacement/repair become an ideal material in the application, these joint to be subjected to repetitive or periodic pressure load (such as ankle joint). In addition to the hydrogel 4 the nature, hydrogel 5 cartilage-like nature, the appearance is similar to the joint of the blade cutting operation for and cartilage with cartilage feeling.

In a closed pressure-proof test in order to quantitatively determine the above-mentioned five kinds of different mechanical properties of compression of the hydrogel. Closed compression resistant test use of tailored polycarbonate fully sheathed case and a porous polypropylene filter drum (20 the hole   m, 20% has the porosity). Using fully sheathed case and the following embodiment 4 the freeze-thaw technique to prepare the hydrogel concentration five has a cylindrical bolt (diameter of 7.1 mm, a thickness of about 3 mm). Then in accordance with the following test steps of the closed compression in a series of stress release experiment. All experimental Instron   5543 tester carried out under control of the computer, machines to 10Hz frequency recording time-displacement-load data. Each experiment using ± 5N or ± 50N load capacity meter the load capacity of the whole tracking (Sensotec). Each step use the 30   m (the 30  m/seconds) speed, the speed equivalent to the 1% tension, until the sample reaches the equilibrium. This is defined as the relaxation rate, when the relaxation rate slow down to 10mN   min^-1 the following, the next step until the completion of the 20 cycles (equivalent to about 20% of the tension). Closed compressive test in the thickness of each sample is determined by mechanical method, by Instron   5543 instrument measuring compression due to the reaction of the sample with respect to the displacement of the bottom of the case. Testing the thickness of the each step for calculating the percentage of the tension.

Five kind of hydrogel compression mechanical properties determined before the adoption of the method of the previous section. Load data sample cross-sectional area for (39.6 mm²) the standard in order to calculate the stress. Each material formulation traces the spot balance stress to the added tension of the drawing. Each step is defined as the coefficient of aggregation of the balanced stress divided by added tension. For each material, aggregation coefficient defined as balance stress-tension data of the linear best slope in the range of. Figure 4 shows the closed compressive the results of the test. All five hydrogel can be closed pressure-proof test and the test that a typical example of two-phase material (such as cartilage) characteristic of the tension relaxation reaction. 6.25 mg/ml and 12.5 mg/ml hydrogel   H-TA the aggregation of low-articular cartilage coefficient than that of the 1 [...] 2 orders of magnitude. 25 mg/ml hydrogel   T-HA coefficient is the aggregation of half of reports the value of articular cartilage. 50 and 100 mg/ml of the hydrogel of   T-HA gathered coefficient in a linear region of less than the reported value of the articular cartilage, at the articular cartilage but is larger than the 15 [...] 20% the value of the tension of the. These data prove that the standard mechanical analysis for the hydrogel can be identification, and evidence the articular cartilage can be generated with similar mechanical properties of the hydrogel.

Based on the above experimental, surprisingly and unexpectedly found that two tyramine cross-linked hyaluronic acid network can produce the adhesion of the hydrogel material, in order to adapt to specific application, the tyramine group through the change in the concentration of cross-linking the hydrogel T-HA can change the hardness and other physical properties (rheology). Even if the network has no (or substantially no) any SO₄^2- group provides charge-charge repulsion force and the elastic resistance of the material, still observe these hydrogel adhesion and elastic. This rather surprising and unexpected results in tissue engineering applications may have in the role of a very positive. Hyaluronic acid is found in the human body existence and generally the height of the non-immune reactive molecule. Therefore, is composed of two tyramine cross-linked hyaluronic acid hydrogel networks may become a very appropriate tissue replacement material implanted into the human body moving, its hardness can be adjusted according to the needs of the application. Because of these material is mainly composed of a has not changed immune reactivity of free hyaluronic acid, people believe that the material may be caused by immune reaction is zero or close to zero. This is and many traditional tissue engineering material with an important advantage compared with, the traditional material in the chemical synthesis due to the harsh reaction conditions or reaction reagent, the application of the in vivo has been impeded, and the material of the final chemical structure has a greater possibility of the induced immune response.

Embodiment 4

Have been developed various preparation method is used in pre-determined three-dimensional shape to prepare or to form a hydrogel, such as the above-mentioned embodiment 3 in the hydrogel. For many tissue engineering applications this is very important, in these applications must be filled artificial tissue material defect or natural tissues of the patient in the cavity.

1st kind of method is to adopt the home position spot forming technique, the hydrogel in this technology, is formed at the original position, that is, the final application of the position and the shape of the structure formed. The method for forming the original site carried out according to the following experimental method. Tyramine-substituted hyaluronan (T-HA) through the in this description of the method for preparing carbodiimide-mediated. In the dialysis to remove unreacted tyramine, EDC, such as the product and NHS in an ideal concentration in PBS after the copolyester (see embodiment 1), will be a small amount of horseradish peroxidase to 1st preparing liquid T-HA formed in solution. The internal solution into the 1st having a specific geometrical shape in the laboratory containers (analog in vivo site). Subsequently, preparation containing very dilute hydrogen peroxide solution 2nd (final concentration of the 0.012% [...] 0.00012%). Subsequently the solution with respect to the 1st and 2nd small volume of the solution had been injected into the container is provided with a 1st solution initial two tyramine cross-linking reaction to produce the hydrogel. Through this technology the hydrogel prepared with the above-mentioned embodiment 3 described in and rheological properties of different hardness, and good preparation of the container and the inner surface profile anastomosis. Because the main reagent (H₂ O₂, hyaluric and peroxidase) is a non-allergic molecule or molecular diffusivity, and because the cross-linking reaction in the metabolic and the temperature of the pH, this technology can be used as a surgical procedures in the surgical site of the patient the operation, thereby generating a defect adaptation hydrogel. The method has the particularly attractive facial reconstruction surgery, the doctor can surgical department the operation of the non-cross-linked (containing peroxidase) solvia T-HA for subcutaneous injection and operation in order to form the ideal facial contour, then small volume through the injection of the hydrogen peroxide solution of hydrogel undergoing a cross-linking.

2nd kind of method is the porous mold technology, with more complex suitable for forming a three-dimensional structure of the hydrogel. First in this technology, according to the required final structure preparing porous form and outline of the hollow mould. For example, if the need to cuboidal hydrogel, a cubic model can be prepared in the inner surface of the body. Mould of traditional porous material can be used for through the traditional technology for preparing or casting, for example plaster, porous or sintered plastics or metal and the like. In a particularly preferred specific embodiment, the mould with a cellulose dialysis membrane preparation. According to the above-mentioned 1st and 2nd solution preparation method, the solution into the 1st multi-hole die hollow of in mold nest. Subsequently, the mold that is filled with very dilute peroxide Spatholobi of no. Macromolecule T-HA and peroxidase because of its molecular size can not be diffused in the porous mold of the die, but very small superoxyde molecular (H₂ O₂) to diffuse into the mould and the presence of the peroxidase reaction occurs generating two tyramine cross-linked. In this method the cross-linked from outside to inside in the hydrogel produce the final shape, and the needed in peroxide bath a certain degree of trial and error in order to determine the optimum or full immersion time. The ordinary technicians in this field has the ability to determine the length of this period of time. Through this kind of the porous mold technology in the context of laboratory experiment has been successfully completed three-dimensional hydrogel preparation.

3rd kind of method is freeze-thaw techniques, the technology is suitable for making a pre-determined height of the complex three-dimensional shape of the hydrogel of this invention, for example the human ear internal recurve. In this technique, a soft or mould for the ductility of the material preparation, such as having a low glass transition temperature of the polymer material, for example below -80 the [...]. Preferably, the mold material having a low glass transition temperature is a silicon gum, such as the glass transition temperature is about -127 the polydimethyl siloxane [...] , of course, other appropriate low glass transition temperature of the silicon gum (for example lower than -80 the [...]) and the other polymer may be also used. The first technical or application of through a traditional (i.e. die forming method, such as method for engraving) preparation of silicon gum (first-selected material), so that the interior of the hydrogel mold nest and the required part of the surface shape, contour and with the volume. The above preparation 1st and 2nd to 1st solution and solution in the inside of the silicon tree jelly moldmold nest. Silicon tree jelly mold subsequently after filling with solid CO₂ (dry ice) contact frozen to about -80 the [...]. Because the 1st solution is primarily water, in the form of a solution solid state ice the inner surface of the mold with the shape and outline. However, glass transition temperature lower than -80 the silicon [...]tree jelly mold is still soft and malleable, it is possible to easily form the 1st solid state ice taken out of the solution. The frozen solution in the 1st expansion occurs, should be the use of an appropriate mechanical equipment to ensure that the silicon tree jelly mold expansion of the solution will not be deformed or expansion. Preferably, to provide access in the mould, when the 1st solution during the freezing process allows the expansion of expansion and release of the solution.

Once the 1st makes the moldsolid state ice the form of solution, in the three-dimensional structure can be small defect or flaw by carving the use of an appropriate tool to repair, and more may be added to liquid 1st solution filling the surface of the cavity, and the liquid forms solid state ice contact instantaneous freezing. Similarly, if it is desired to ensure that consistent temperature and 1st solution to ensure that all of the freezing of the material, dry ice surface can be put back into the ice. Once the three-dimensional shape of the ice to form a perfect, will it is submerged in the liquid peroxide solution while the initial frozen water from the thawing and two tyramine cross-linked. The crosslinking reaction can be rapidly dynamics, this kind of method is feasible. The center of the hydrogel when forming the last remaining frozen water melting completion of cross-linking that, because of forming the hydrogel substantially transparent, it is easy to observe completion of the cross-linked.

According to this freeze-thaw technique has been very successful experiments the shape of the human ear to produce a solid hydrogel of the present invention. This kind of method can be formed by other structure, such as intervertebral disc, meniscofemoral, and the like, the technical personnel in the field are obvious. In the freeze-thaw technique should be noted that in, die material critical glass transfer temperature -80 the is roughly [...] according to solid CO₂ (dry ice) the temperature of the surface of the selected, so as to ensure that the generated solution 1st solid state ice form mould material will not become brittle. However, if the use of CO₂ freezing materials other than, of the mould material of appropriate critical glass transfer temperature can be adjusted accordingly.

Generating the above-mentioned three methods of the hydrogel, solution containing peroxidase and T-HA 1st, and 2nd solution containing peroxide. Although can be exchanged in the 1st and 2nd solution of peroxidase and peroxide, but the peroxide and adding T-HA 1st solution and non-optimized. This is because once the peroxide, peroxidase and T-HA mixed, to begin rapidly T-HA form a cross-linked macromolecular network. If peroxidase (peroxidase is macromolecule) is not uniformly distributed within T-HA, peroxidase may not be able to or substantially to the hydrogel by forming the hole to spread, it is difficult in the whole T-HA/peroxide solution are generated in a cross-linked. The result may lead to non-uniformity of T-HA and/or does not completely cross-linked and non-homogeneous hydrogel. On the contrary, relatively small peroxide molecule (hydrogen peroxide than water of one oxygen atom) can be relatively easily through the hydrogel pore structure to carry out diffusion, of the hydrogel structure is generated.

Furthermore, the large molecular weight of peroxidase with the similar T-HA in the porous mould, the mould only to the small molecular weight peroxide permeability, peroxide can be through mould and new production large molecular network (in other words hydrogel) a relaxed and diffusion occurs. For these reasons, preferably, in a 1st solution T-HA peroxidase and uniform distribution, provided separately in the 2nd solution of peroxide.

4th kind of method is interactive spray layers or brushes the coating duplicate technology. Preparation according to the above-mentioned method and comprising a peroxidase enzyme and 1st T-HA solution. However, not only the 2nd solution containing the above-mentioned peroxide, and contains the T-HA 1st solution of the same concentration. 1st thin layer of the solution is then coated on the desired site (primary site) 2nd subsequent thin layer of the solution on the cover. This step is repeated continuously alternating 1st and 2nd solution spray to the layer of filling completed defect or using site. The 1st and 2nd very thin alternating layers of the solution to promote the completion of the two tyramine cross-linked, the height of the to ensure the consistency of the final hydrogel, and the water gel composed of two solution has an ideal initial T-HA concentration of rheology nature of the decision. Ideal liquid layer is very thin, in 1st solution layer in order to ensure that the generated free radical peroxidase can completely penetrate into the adjacent layer and complete the 2nd solution of cross-linking, the cross-linking does not depend on the proliferation of peroxidase to the 2nd solution layer (see above). In the two solution containing T-HA thereby ensuring that the final hydrogel T-HA concentration of. In experiments in the laboratory have been completed the technical intercrescence into the contour and a large amount of filling a uniform hydrogel. When needing to produce thin of tyramine cross-linked HA layer, the technology is highly suitable, such as in the surface of the bare joint osteoarthritis, the transplant site of the patient is almost without a healthy cartilage.

All of the above four kinds of technology on two tyramine cross-linked hyaluronic acid to describe, however, should be aware that within the scope of the present invention other combinations of (other dihydroxy divinylbenzene macromolecule, such as gathers the carboxylic ether , polyamine, polyethylene hydroxybenzotriazole elements and their copolymer) can be through the above-mentioned technology moulding.

Embodiment 5

The RAT cartilage cells T-HA buried (cross-linked) in the hydrogel in order to test the viability of cross-linked in the reaction. The live cells into solution with the 1st T-HA and peroxidase common dispersion, then the 2nd solution of peroxide containing initial two tyramine cross-linked, thus separating the cartilage cells are suspended in the embodiment 2 is described in the 1.7% and 4.7% of hydrogel T-HA. Cartilage cells are buried 1.7% and 4.7% T-HA hydrogel is uniformly distributed in the cartilage cell, and gel visual transparent, visibility of the entire gel has. The cross-linked hydrogel-forming glucose utilization as an indicator of cell viability after, glucose consumption because cartilage cell to great, in less than 24 hours to make the inner surface of the glucose culture medium. Embedded in the hydrogel T-HA with cartilage cells in monolayer culture of the same cartilage cells in 24 hours have substantially the same glucose consumption condition (Figure 5). This situation persists as long as 7 days, the cell is alive and by metabolic active. Culture-based glucose hexose kinase analysis through the standard test.

With the cartilage cells and cartilage tissue of freezing of the hydrogel T-HA part also the fluorescent imaging. Hydrogel framework and HA cartilage matrix of the sample through the biotin-labeled binding protein HA imaging fluorescent dyeing reagent (b-HABP), DAPI for the cell nucleus of dyeing imaging. The purification of reagents b-HABP cartilage aggrecan (containing only G1 domain) and is connected with the protein preparation, the reagent recognition does not reversibly with the natural HA peice combined, under normal conditions these segments in the cartilage aggrecan and is connected with the protein. Display the results of the staining of the hydrogel T-HA b-HABP comparison cartilige the dyeing effect is stronger, because of the organization of hyaluronic acid have been combined in the natural aggrecan and is connected with the protein. In hydrogel T-HA frame and suspension of the cartilage there is no obvious between the substrates are respectively means that the tight integration. These results demonstrate that the cross-linking reaction in the hydrogel in the feasibility of maintaining cartilage cell activity, hydrogel closely and integrated into the existing capacity of the cartilage matrix, both the application of the cartilage repair is very favorable. T-HA results also prove sufficient number of fragments in the chemical is maintained unchanged, and the synthesis of the new home position spot aggrecan and connecting protein binding. The results also confirmed that oxygen, carbon dioxide, glucose and insulin the hydrogel T-HA of this invention in the spread to a certain rate, the rate of metabolism of the cartilage cells limit does not occur, this is not only very important for the cartilage replacement material, but also for other applications such as glucose and inductor design is very important for the development of artificial kidney.

In order to the cells such as chondrocytes by embodiment 4 in the refrigerating/melt technology to complex shape of the molding in the hydrogel of the organization, it is hoped that in the standard enzyme actuation the cells of the cross-linking reaction in the presence of a frozen solution, for example containing 10% dimethyl sulfoxide (DMSO)/ 90% fetal bovine serum (FBS) cell freezing solution. This point has been in the laboratory through the embodiment in 3 T-HA described in all of the hydrogel formulations to be confirmed. Can be added directly to the containing 90% FBS solution is also proved that the reaction can comprise biologically active factor, such as a growth factor, hormone and control cell differentiation factor, FBS because these factor is the normal component.

Although the above specific embodiment of the preferred embodiment, it should be clear that, in the attached not deviate from the spirit and of the present invention under the situation of range, to the present invention can be various changes or correction.