JP6846103B2 - How to make a complex of phosphorylated chitin and titanium, and the complex - Google Patents

Description

The present invention relates to a method for producing soluble phosphorylated chitin or soluble phosphorylated collagen, a method for producing a complex of phosphorylated chitin or phosphorylated collagen and titanium, a method for producing a polyurethane foam, a complex, soluble. The present invention relates to a method for producing phosphorylated collagen and a method for producing a complex of phosphorylated collagen and titanium.

Bio-derived polymer compounds (hereinafter, also simply referred to as “biopolymers”) contain many compounds that have unique properties but are chemically difficult to produce. In addition, biopolymers are materials that are abundant in nature and are often harmless or harmless to living organisms. Therefore, medical applications of biopolymers are being studied.

For example, it is known that when a complex is formed between a phosphorylated protein and titanium or a titanium alloy, titanium or the titanium alloy can be firmly bonded to bone in a short period of time (Patent Documents). 1 etc.).

As an example of a method for phosphorylating a biopolymer, a method for phosphorylating purulan by mixing and reacting an aqueous solution of purulan and an alkaline compound in water and a phosphorylating reagent is known. (Patent Document 2, etc.). According to Patent Document 2, the phosphorylated and solubilized pullulan has a high affinity for living tissue and also has an adsorptive property for a metal which is a prosthetic material for living hard tissue.

However, many biopolymers are often insoluble or sparingly soluble in both water and solvents commonly used in the medical field. Examples of such insoluble or sparingly soluble biopolymers (hereinafter, insoluble and sparingly soluble are collectively referred to as “insoluble”) constitute the exoskeleton of shrimp, crab, and insect (particularly beetle). It contains chitin and collagen, which is a bioprotein. 6M guanidine hydrochloride solution and 6M urea solution are known as solutions for dissociating and dissolving proteins, but chitin and collagen dissolve only a few percent in both 6M guanidine hydrochloride solution and 6M urea solution. ..

In order to advance the medical application of these insoluble biopolymers, the development of a method for solubilizing the biopolymer is required. For example, chitin is known to dissolve in a weak acid by artificially deacetylating it into chitosan (Patent Document 3 and the like). Further, it is known that collagen is solubilized in water by cross-linking with an alkyldiimidate divalent cross-linking reagent having imide ester groups at both ends of the methylene chain (Patent Document 4, etc.).

Further, as a prosthetic material for a biological hard tissue, a polyurethane foam coated with apatite or a material obtained by sintering a polyurethane foam containing titanium powder or the like is known (Patent Documents 5, 6 and the like). ).

Japanese Unexamined Patent Publication No. 2014-004327 Japanese Patent No. 5706579 Special Table 2005-500410 Japanese Unexamined Patent Publication No. 07-097454 Japanese Patent No. 5553596 Japanese Patent No. 5634042

Kuboki et al., "Calcified Honeycomb-shaped Collagen Maintains its Geometry in Vivo and Effectively Induces Vasculature and Osteogenesis," Nano Biomedicine, 2009, Vol.1 (2), pp. 85-94

However, it has been clarified that chitosan obtained by a method such as Patent Document 3 has strong immunogenicity (antigenicity) unlike chitin. Therefore, the medical application of chitosan (for example, application to materials to be implanted in the living body) is limited. Further, collagen solubilized by a method such as Patent Document 4 often has its properties changed due to chemical modification or the like, and it is unclear whether or not it can be used medically as it is.

On the other hand, as described in Patent Documents 1 and 2, phosphorylated biopolymers are water-soluble and often have the properties of the original biopolymers without impairing them. , A compound that can be expected to be applied medically. In Patent Document 1, a phosphorylated protein is extracted in a living body to obtain a phosphorylated biopolymer. On the other hand, in order to apply a wider variety of biopolymers medically, it is desired to develop a technique for artificially phosphorylating and solubilizing biopolymers. In Patent Document 2, phosphorylated pullulan is obtained by dissolving water-soluble pullulan in water and phosphorylating it. On the other hand, it is desired to develop a technique for phosphorylating and solubilizing not only water-soluble compounds but also insoluble biopolymers.

The present invention has been made in view of this point, and an object of the present invention is to provide a method for phosphorylating and solubilizing insoluble biopolymers, particularly chitin and collagen. Further, the present invention is to apply the above-mentioned insoluble biopolymer solubilized by phosphorylation to a composite with titanium, a polyurethane foam, or the like to provide a wide range of medical and industrial uses. The purpose.

INDUSTRIAL APPLICABILITY The present invention relates to the following methods for producing soluble phosphorylated chitin or soluble phosphorylated collagen, a method for producing phosphorylated chitin or a composite of phosphorylated collagen and titanium, a method for producing a polyurethane foam, and phosphorylation. The present invention relates to a method for producing collagen and a method for producing a complex of phosphorylated collagen and titanium.

[1] A step of dispersing chitin or insoluble chitin in an aprotic solvent, a step of phosphorylating the dispersed chitin or the dispersed insoluble chitin in the presence of a catalyst, and a phosphorylated chitin. Alternatively, a method for producing soluble phosphorylated chitin or soluble phosphorylated collagen, comprising the step of separating the phosphorylated insoluble collagen.
[2] The method for producing soluble chitin or soluble phosphorylated collagen according to [1], wherein the aprotic solvent is a single solvent of hexane.
[3] The aprotic solvent is a mixed solvent of hexane and dimethylformamide, and the volume ratio of hexane and dimethylformamide (hexane: dimethylformamide) in the mixed solvent is in the range of 25:75 to 100: 0. The method for producing soluble phosphorylated chitin or soluble phosphorylated collagen according to [1], which is (however, does not contain 100: 0).
[4] The method for producing soluble phosphorylated chitin or soluble collagen according to any one of [1] to [3], wherein the separation step is dialysis and centrifugation in this order with respect to water.
[5] The method according to [4], wherein in the separation step, after the dialysis, a component insoluble in water remaining in the dialysis internal solution is dissolved in an organic solvent, and alcohol is further added to perform dialysis against water. A method for producing soluble phosphorylated chitin or soluble phosphorylated collagen.
[6] The above phosphorylation step is performed so that the temperature of the dispersion liquid in which the insoluble collagen is dispersed is 40 ° C. or higher and 50 ° C. or lower, and further, the remaining insoluble collagen is redispersed after the separation step. In the step and in the presence of a catalyst, the temperature of the dispersion containing the redispersed insoluble collagen is set to 60 ° C. or higher and 90 ° C. or lower, and the redispersed insoluble collagen is phosphorylated to obtain phosphorylated gelatin. The method for producing soluble phosphorylated chitin or soluble collagen according to any one of [1] to [5], wherein the step and the step of separating the phosphorylated gelatin are performed.
[7] The solubility according to any one of [1] to [6], further comprising a step of separating the phosphorylated chitin or the phosphorylated insoluble collagen according to its properties after the separation step. A method for producing phosphorylated chitin or soluble collagen.
[8] The soluble step according to [7], wherein the step of separating the phosphorylated chitin or the phosphorylated insoluble collagen according to its properties is a step of performing affinity chromatography using titanium beads as a stationary phase. A method for producing phosphorylated chitin or soluble collagen.
[9] Phosphorylation comprising a step of contacting a solution containing soluble chitin or soluble collagen phosphorylated produced by the method according to any one of [1] to [8] with a titanium substrate. A method for producing a complex of chitin or phosphorylated collagen and titanium.
[10] The phosphorylated chitin or phosphorus according to [9], further comprising a step of immersing the titanium base material in contact with the solution containing the soluble chitin or soluble collagen in the calcification solution. A method for producing a complex of collagen oxide and chitin.
[11] A composite having a base material made of titanium or a titanium alloy and a phosphorylated chitin-containing layer or a phosphorylated collagen-containing layer that covers at least a part of the surface of the base material.
[12] The complex according to [11], wherein the phosphorylated chitin-containing layer or the phosphorylated collagen-containing layer contains hydroxyapatite.
[13] A step of preparing a mixed solution containing soluble phosphorylated chitin, polyols and polyisocyanates produced by the method according to any one of [1] to [8], and adding a foaming agent to the mixed solution. A method for producing a polyurethane foam, which comprises the steps of mixing, stirring and mixing.
[14] A step of reacting polyols with polyisocyanates to produce a prepolymer having an isocyanate group at the terminal, and a soluble phosphorylated chitin produced by the method according to any one of [1] to [8]. A method for producing a polyurethane foam, which comprises a step of preparing a mixed solution containing the prepolymer, excess water and polyols, and a step of mixing the mixed solution with a foaming agent, stirring and mixing.
[15] A step of dispersing soluble collagen in a phosphate buffer having a concentration of 0.5 mol / L or more and 1.0 mol / L or less, and phosphorylation of the dispersed soluble collagen in the presence of a catalyst. A method for producing phosphorylated collagen, which comprises a step of separating the phosphorylated soluble collagen.
[16] The method for producing phosphorylated collagen according to [15], further comprising a step of separating the phosphorylated soluble collagen according to its properties after the separation step.
[17] The method for producing phosphorylated collagen according to [16], wherein the step of separating the phosphorylated soluble collagen according to its properties is a step of performing affinity chromatography using titanium beads as a stationary phase.
[18] A composite of phosphorylated collagen and titanium, which comprises a step of bringing a solution containing soluble collagen produced by the method according to any one of [15] to [17] into contact with a titanium base material. How to make a body.

According to the present invention, insoluble biopolymers, particularly chitin and collagen, can be phosphorylated and solubilized. Further, according to the present invention, the above-mentioned insoluble biopolymer solubilized by phosphorylation is applied to a composite with titanium, a polyurethane foam, or the like to provide a wide range of medical and industrial uses. It will be possible.

FIG. 1 is an infrared absorption spectrum measured for a chitin sample obtained by phosphorylation and solubilized in Example 1. FIG. 2 is an infrared absorption spectrum of the phosphorylated and solubilized chitin sample and the non-phosphorylated chitin obtained in Example 1. FIG. 3 is an infrared absorption spectrum of the collagen sample obtained in Example 3 which was solubilized by phosphorylation and the collagen sample which was not phosphorylated. FIG. 4 is a chromatogram showing the elution pattern of phosphorylated chitin with respect to the column packed with titanium particles in Example 4. FIG. 5 is a chromatogram showing the elution pattern of phosphorylated collagen with respect to the column packed with titanium particles in Example 5. FIG. 6 is a chromatogram showing the elution pattern of collagen that has not been phosphorylated with respect to the column packed with titanium particles in Example 5. FIG. 7A and a partially enlarged view of FIG. 7B are tissue specimens from rats in which the implants of Example 6 were implanted. FIG. 7C and partially enlarged FIG. 7D are tissue specimens from rats in which the implant of Comparative Example in Example 6 was implanted. FIG. 8A and partially enlarged FIG. 8B are photographs taken by a scanning electron microscope (SEM) of the surface of the complex of phosphorylated chitin and titanium before the hydroxyapatite precipitation treatment. FIG. 8C and partially enlarged FIG. 8D are SEM photographs of the surface of the complex of phosphorylated chitin and titanium in which hydroxyapatite is precipitated. FIG. 9 is a graph showing the average mass of the polyurethane foam before precipitating hydroxyapatite in Example 9, and the masses of the test piece 1, the test piece 2, and the test piece 3.

[First Embodiment]
The first embodiment of the present invention relates to a method for producing soluble chitin and soluble collagen. In this embodiment, a step of dispersing chitin or collagen in a specific dispersion medium, a step of phosphorylating the dispersed chitin or collagen in the presence of a catalyst, and a step of separating the phosphorylated chitin or collagen. And, including. The present embodiment may further include a step of separating phosphorylated chitin or collagen according to its properties. The term "soluble" means a state in which the solubility in water or an organic solvent is higher than that of living-derived chitin or collagen. In this embodiment, soluble phosphorylated chitin or collagen is produced by phosphorylating dispersed chitin or collagen at least partially in the presence of a catalyst. The phosphorylated collagen means a protein in which three peptide chains having a so-called collagen sequence in which glycine repeats every three bases form a spiral structure, at least partially phosphorylated. To this extent, collagen constituting phosphorylated collagen may be denatured into gelatin or the like.

(Step of dispersing chitin in a specific dispersion medium)
In this step, chitin or collagen is dispersed in a specific dispersion medium. The specific dispersion medium is an aprotic solvent in the case of chitin or insoluble collagen, and a phosphate buffer having a concentration of 0.5 mol / L or more and 1.0 mol / L or less in the case of soluble collagen. is there.

The aprotic solvent used for dispersing chitin or insoluble collagen means a solvent having no proton donating property. Examples of aprotic solvents include diethyl ether, diisopropyl ether, tetrahydrofuran, pentane, n-hexane, cyclohexane, heptane, benzene, toluene, xylene, dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, dimethylformamide, Includes dimethyl sulfoxide, acetonitrile, acetone and tetrahydrofuran.

Of these, the aprotic solvent is preferably a single solvent of hexane or a mixed solvent of hexane and dimethylformamide. When the aprotic solvent is a mixed solvent of hexane and dimethylformamide, the volume ratio of hexane to dimethylformamide (hexane: dimethylformamide) in the mixed solvent is in the range of 25:75 to 100: 0 (however, however. It does not include 100: 0).

The aprotic solvent can be selected depending on the properties of the phosphorylated chitin or phosphorylated collagen to be obtained. For example, when the aprotic solvent is a single solvent of hexane, most of the resulting phosphorylated chitin or phosphorylated collagen exhibits water-soluble properties. On the other hand, when the aprotic solvent is a mixed solvent of hexane and dimethylformamide and the volume ratio of hexane to dimethylformamide is in the range of 25:75 to 50:50, the phosphorylated chitin or phosphorus obtained is obtained. Oxidized collagen is a mixture of those exhibiting water-soluble properties and those exhibiting properties that are insoluble in water but soluble in an organic solvent (for example, dimethylformamide).

The phosphate buffer having the above concentration of 0.5 mol / L or more and 1.0 mol / L or less used for dispersing soluble collagen was prepared, for example, with sodium dihydrogen phosphate and disodium hydrogen phosphate. It can be a phosphate buffer solution or a phosphate buffer solution prepared by disodium hydrogen phosphate and potassium dihydrogen phosphate. At this time, the pH of the phosphate buffer is preferably less than 7, and from the viewpoint of facilitating pH adjustment, a phosphate buffer prepared with disodium hydrogen phosphate and potassium dihydrogen phosphate is preferable. ..

Dispersion can be carried out by a known method. For example, the chitin or collagen can be dispersed by putting the dried chitin or collagen powder and the above-mentioned specific dispersion medium in a closed container and stirring or shaking the mixture. At this time, if it can be visually confirmed that the aprotic solvent is turbid due to the dispersion of chitin or collagen, it may be determined that the chitin or collagen is sufficiently dispersed.

(Step of phosphorylating dispersed chitin or collagen in the presence of catalyst)
A method catalyzed by a phosphorylating enzyme (kinase), which is known as a method of phosphorylating a polymer in a living body, is generally difficult due to the high specificity of this type of enzyme. Therefore, a chemical phosphorylation method using a non-enzymatic catalyst has been pursued, but a sufficiently effective method has not yet been developed.

On the other hand, the phosphorylation of dispersed chitin or collagen in the present invention can be carried out by a method using a known catalyst, which is known as a method for phosphorylating a biopolymer. Known methods for phosphorylating biopolymers include an enzymatic method using an enzyme as the catalyst, and a chemical method using a phosphorylation reagent and a catalyst suitable for phosphorylation thereof.

Examples of phosphorylating enzymes that have the potential to phosphorylate chitin or collagen include the highly specific cyclic AMP-dependent protein kinase (EC 2.7.11.37).

The temperature at the time of stirring in the above enzymatic method is preferably around the active temperature of the above enzyme.

Examples of the chemical method include a method in which a known phosphorylation reagent and a catalyst that can be used for phosphorylation by the known phosphorylation reagent are added to the dispersion and stirred.

The phosphorylation reagent that can be used for phosphorylation of chitin or collagen is preferably phosphorus pentoxide. Other examples of the above-mentioned phosphorylation reagents include sodium primary phosphate (sodium dihydrogen phosphate), sodium dibasic phosphate (disodium hydrogen phosphate), sodium tertiary phosphate (trisodium phosphate), and tripolyline. Sodium acid, sodium pyrophosphate, sodium metaphosphate, potassium primary phosphate (potassium dihydrogen phosphate), potassium dibasic phosphate (dipotassium hydrogen phosphate), potassium tertiary phosphate (tripotassium phosphate), tripolyline Includes potassium acid, potassium trimetaphosphate, and phosphorus oxychloride.

The amount of the phosphorylation reagent may be appropriately adjusted according to the desired properties of the obtained phosphorylated chitin or phosphorylated collagen. From the viewpoint of sufficiently solubilizing chitin or collagen and reducing the effect on the living body, the phosphorylation reagent has a phosphate ion content of 10% by mass or more and 200% by mass or less with respect to chitin or collagen. It is preferable that the amount is added.

Examples of catalysts that can be used for phosphorylation of chitin or collagen by the above chemical methods include methanesulphonic acid.

The temperature at the time of stirring in the above chemical method may be a temperature at which denaturation of chitin or collagen does not occur and phosphorylation of chitin or collagen sufficiently occurs. From the viewpoint of sufficiently phosphorylating and solubilizing chitin or collagen, the temperature at the time of stirring is preferably 40 ° C. or higher and 50 ° C. or lower.

When phosphorylating insoluble collagen, not all collagen is solubilized at the above temperatures, and some collagen may remain insoluble. The unsolubilized collagen is further dispersed (redispersed) in an aprotic solvent by the above method, and phosphorylated at 60 ° C. or higher and 90 ° C. or lower, preferably 70 ° C. to obtain phosphorylated gelatin by the above method. Further, by separating by the method described later, it can be recovered as phosphorylated gelatin.

The stirring time in the above chemical method may be a time during which phosphorylation of chitin or collagen sufficiently occurs, and may be, for example, 2 hours or more and 5 hours or less.

(Step to separate phosphorylated chitin or collagen)
The chitin or collagen phosphorylated by the above method is soluble in water or an organic solvent. Therefore, by separating the phosphorylated chitin or collagen by the above method into water or an organic solvent, a solution containing soluble chitin or phosphorylated collagen can be obtained.

Separation can be performed by a known method. Examples of separation methods include dialysis and centrifugation, and it is preferable to perform dialysis and centrifugation on water (for example, distilled water) in this order. From the viewpoint of improving the accuracy of separation, it is preferable to perform dialysis a plurality of times until there is no change in the electrical conductivity of the external liquid after dialysis.

From the viewpoint of further improving the accuracy of separation, it is preferable to dissolve the sediment obtained after dialysis in an organic solvent (for example, dimethylformamide), and then add alcohol to further perform dialysis with water. Examples of the above alcohols include methanol and ethanol.

(Step of separating phosphorylated chitin or collagen according to its properties)
The chitin or collagen phosphorylated by the above method includes a chitin derivative or a collagen derivative having various properties, probably due to differences in the amount of phosphoric acid groups bonded and the distance between the phosphoric acid groups. The present embodiment may include a step of separating phosphorylated chitin or phosphorylated collagen having these different properties according to their properties. For example, affinity chromatography can be used to separate compounds that bind or adsorb to the stationary phase of a column from the phosphorylated chitin or phosphorylated collagen.

As the stationary phase of the column, a substance to which phosphorylated chitin or phosphorylated collagen having the property to be obtained binds or is adsorbed may be used. For example, by using titanium beads as the stationary phase, phosphorylated chitin or phosphorylated collagen having a titanium-binding property can be separated and obtained.

It is known that some phosphorylated biopolymers have an ability to bind to titanium (see, for example, Patent Document 1 and Patent Document 2). Titanium is also used as a bone reconstruction material for artificial bones, artificial joint shafts or artificial tooth roots. Therefore, the phosphorylated chitin or phosphorylated collagen having the property of binding or adsorbing to titanium separated by the method according to the present embodiment can be bound to titanium and applied to a bone reconstruction material (described later). See second and third embodiments).

(effect)
Since the soluble phosphorylated chitin or soluble phosphorylated collagen produced in this manner is soluble in water or an organic solvent, it can be easily mixed or reacted with other substances, administered to a living body, or the like. It is possible and suitable for industrial and medical applications. Further, since this soluble phosphorylated chitin or soluble collagen has without impairing the properties of the original chitin or collagen, it has little adverse effect on the living body and is suitable for medical application.

[Second Embodiment]
A second embodiment of the present invention relates to a complex of phosphorylated chitin or phosphorylated collagen and titanium, and a method for producing such a complex. The method according to the present embodiment includes a step of bringing a solution containing soluble chitin or soluble phosphorylated collagen produced by the method according to the first embodiment into contact with a titanium base material. The phosphorylated chitin or the composite of phosphorylated collagen and titanium produced by the method according to the present embodiment is a base material made of titanium or a titanium alloy, and phosphorylated chitin that covers at least a part of the surface of the base material. It has a containing layer or a phosphorylated collagen-containing layer.

From the viewpoint of more sufficiently covering the phosphorylated chitin-containing layer or the phosphorylated collagen-containing layer with the titanium base material, only those having the property of binding or adsorbing to titanium are separated from the soluble chitin or phosphorylated collagen. It is preferable that the product is made.

The titanium base material may be a base material having titanium or a titanium alloy at least on its surface. The shape of the base material can be appropriately selected depending on the use of the composite. For example, when the composite produced in the present embodiment is applied to a bioimplant, examples of the shape of the base material include columnar (rod-like), plate-like, sheet-like, block-like, wire-like, fibrous, and powder-like. The condition etc. are included. When the complex produced in the present embodiment is applied to a cell culture substrate, examples of the shape of the substrate include a mesh shape (including a non-woven fabric (titanic web: TW)), a flat plate shape, and the like. ..

As the solution containing soluble phosphorylated chitin or phosphorylated collagen, the solution obtained in the step of separating phosphorylated chitin or phosphorylated collagen in the first embodiment is used as it is, or concentrated or diluted. Alternatively, a phosphorylated chitin or phosphorylated collagen obtained by drying the solution obtained in the separation step may be dissolved in another solvent.

The contacting step can be performed by a known method. For example, the titanium substrate may be immersed in the solution containing the soluble chitin or soluble collagen, or the solution containing the soluble chitin or soluble collagen may be applied to the surface of the titanium substrate. You may.

The contacting step can be performed by, for example, the following steps. All the operations in the following steps are performed in a clean bench under sterile conditions. The soluble phosphorylated chitin or phosphorylated collagen is dissolved in a physiological buffer solution (for example, Dalbecco physiological buffer solution) so that the final concentration is 0.1 to 1.0%, and then sterile filtered. Prepare a phosphorylated chitin solution or a phosphorylated collagen solution. The phosphorylated chitin solution or phosphorylated collagen solution is applied to the surface of the base material, or the base material is immersed in the phosphorylated chitin solution or phosphorylated collagen solution, and the phosphorylated chitin or phosphorylation is carried out on the surface of the base material. Adsorbs collagen. Then, after removing the excess liquid by suction, the surface of the base material is dried. The base material (titanium-phosphorylated chitin complex or titanium-phosphorylated collagen complex) on which the phosphorylated chitin or phosphorylated collagen is adsorbed is stored in a sterile container in a dry state at 10 ° C. or lower.

The phosphorylated chitin-containing layer or the phosphorylated collagen-containing layer may contain other components in addition to the phosphorylated chitin or the phosphorylated collagen, depending on the intended use. In this case, in order to more fully exert the binding property of phosphorylated chitin or phosphorylated collagen to bone, the amount of phosphorylated chitin in the phosphorylated chitin-containing layer or the phosphorylated collagen in the phosphorylated collagen-containing layer The amount is preferably 80% by mass or more.

(effect)
In the composite thus produced, the surface of the titanium base material is coated with phosphorylated chitin or phosphorylated collagen having high affinity for both titanium and various cells contained in bone. Therefore, when the complex produced by the method according to the present embodiment is used as a bioimplant (for example, artificial bone, artificial tooth root, etc.), it can be firmly bonded to the bone in a short period of time. In addition, when the complex produced by the method according to the present embodiment is used as a cell culture base material, various cells contained in bone can be suitably cultured.

[Third Embodiment]
A third embodiment of the present invention relates to a complex of phosphorylated chitin and titanium or a complex of phosphorylated collagen and titanium, and another method for producing such a complex. The present embodiment includes the step of binding a cell-adherent protein or a partial peptide thereof to the phosphorylated chitin or phosphorylated collagen in the method according to the second embodiment.

Examples of the cell adhesion proteins include proteins having an RGD sequence involved in cell adhesion on the molecular surface, and synthetic peptides containing the same sequence. The RGD sequence is a sequence consisting of 3 residues of Arg-Gly-Asp, which is _{recognized by α V} β ₃ integrin of cells and specifically binds to it. Examples of proteins having an RGD sequence on the molecular surface include osteopontin (OPN), bone sialoprotein (BSP) and dentin matrix protein-1 (DMP1).

The cell-adherent protein can be bound to the phosphorylated chitin or phosphorylated collagen by a known protein cross-linking agent. For example, if a protein cross-linking agent is appropriately selected, the deacetylated and exposed amino group of the phosphorylated chitin can be bound to the carboxyl group of the cell adhesion protein. Further, if the protein cross-linking agent is appropriately selected, the amino group generated by the fragmentation of the phosphorylated collagen and the like can be bonded to the carboxyl group of the cell-adherent protein.

The binding can be carried out by a known method. For example, the phosphorylated chitin or phosphorylated collagen and the cell-adherent protein may be crosslinked with a protein cross-linking agent.

As the protein cross-linking agent, a wide variety of reagents that have been developed can be used depending on the purpose (see, for example, the catalog of Funakoshi Co., Ltd.). Among them, examples of reagents for cross-linking different reactive groups between two molecules, for example, an amino group and a sulfidyl group, include maleimide-propyloxy-succiniimide and the like.

(effect)
In the composite thus produced, the phosphorylated chitin or phosphorylated collagen that coats the surface of the titanium substrate is further bound to the cell-adherent protein. Therefore, the cell-adherent protein can further promote bone formation between the titanium base material and the bone, and the titanium base material can be bound to the bone in a shorter period of time and more firmly. According to the findings of the present inventors, the composite produced by the method according to the present embodiment can bond to bone at a speed nearly 100 times faster than that of a titanium base material that does not form the composite. it can.

[Fourth Embodiment]
A fourth embodiment of the present invention relates to a composite of phosphorylated chitin and titanium in which hydroxyapatite is precipitated on the surface, and a method for producing such a composite. The present embodiment includes a step of immersing a titanium substrate contacted with a solution containing soluble phosphorylated chitin by the method according to the second embodiment or the third embodiment in a calcification solution. By this step, hydroxyapatite can be bound to phosphorylated chitin or phosphorylated collagen that coats the surface of the complex, and hydroxyapatite can be precipitated.

The calcification solution can be a solution containing phosphate ions and calcium ions in a semi-stable concentration, for example, Kubogi et al. (Non-patent) containing 15 mM / L calcium ions and 9 mM / L phosphate ions. It can be the solution described in Document 1).

The step of immersing in the calcification solution can be performed by, for example, the following step described in Non-Patent Document 1. 15 mM / L calcium chloride (CaCl ₂ ), 9 mM / L dipotassium phosphate (K ₂ HPO4), 0.7 M / L sodium chloride (NaCl) and 0.02 M / L sodium hydrogen carbonate (NaHCO ₃ ) Prepare a calcification solution containing. The composite obtained by the method according to the second embodiment or the third embodiment in the calcification solution whose pH is adjusted to 6.01 to 6.05, preferably 6.01 by bubbling carbon dioxide gas. The body is immersed and incubated at 20 ° C. to 50 ° C., preferably 37 ° C. for 12 to 48 hours, preferably 48 hours. Then, the titanium base material is taken out, washed with desalted water, and freeze-dried. The lyophilized titanium substrate is reimmersed in the new calcification solution and incubated every other day for 1-2 weeks, changing the calcification solution. Then, the titanium base material is taken out, washed with desalted water, and freeze-dried. The titanium substrate on which the hydroxyapatite is precipitated thus obtained is stored in a sterile container in a dry state at 10 ° C. or lower.

(effect)
In the titanium base material produced in this manner in which hydroxyapatite is precipitated on the surface, hydroxyapatite having a high affinity for a living body is evenly precipitated. Therefore, hydroxyapatite can further promote bone formation between the titanium base material and the bone, and the titanium base material can be firmly bonded to the bone in a shorter period of time.

[Fifth Embodiment]
A fifth embodiment of the present invention relates to a method for producing a foam material. The present embodiment includes a step of producing a polyurethane foam containing the soluble phosphorylated chitin obtained in the first embodiment, and a step of producing a foam material. The present embodiment may further include a step of immersing the polyurethane foam in a calcification solution.

(Process for producing polyurethane foam containing phosphorylated chitin)
In the step of producing the polyurethane foam containing phosphorylated chitin, the polyurethane foam can be produced by a conventional method including a one-shot method and a prepolymer method.

The one-shot method includes a step of preparing a mixed solution containing the above-mentioned phosphorylated chitin, polyols and polyisocyanates, and a step of mixing the foaming agent with the mixed solution, stirring and mixing. In the steps of stirring and mixing, the raw materials are reacted by a urethanization reaction, a cross-linking reaction or the like to form urethane, and foam by a foaming reaction. At this time, for example, in the step of preparing the mixed solution, the phosphorylated chitin is added to the polyols in advance and stirred to prepare the polyols in which the phosphorylated chitin is dispersed, and the polyol in which the phosphorylated chitin is dispersed. And polyisocyanates may be mixed.

The prepolymer method is a step of reacting polyols with polyisocyanates to produce a prepolymer having an isocyanate group at the terminal, and a mixed solution containing the phosphorylated chitin, the prepolymer, excess water and polyols. A step of preparing the above, and a step of mixing the foaming agent with the mixed solution, stirring and mixing the mixture. In the steps of stirring and mixing, the prepolymer and the polyols react with each other by a urethanization reaction, a cross-linking reaction or the like to form urethanization, and foam by a foaming reaction. At this time, for example, in the step of preparing the mixed solution, the phosphorylated chitin is added to the excess water and the polyols, and the mixture is stirred to prepare the polyols in which the phosphorylated chitin is dispersed, and the phosphorylation thereof is performed. The above-mentioned prepolymer may be mixed with the polyols in which chitin is dispersed.

Examples of the above-mentioned polyols include polyether polyols such as polyoxyethylene diol, polyoxyethylene triol, polyoxyethylene tetrol, polyoxyethylene hexol and polyoxyethylene octol, and adipates such as poly (butylene adipate) diol. Polyester-based polyols and polyester-based polyols containing caprolactone-based polyols such as poly-ε-caprolactone diols, polyoxyalkylene polyols such as polyethylene glycol, polypropylene glycol and propylene oxide adducts of glycerin, and polycarbonates such as poly (hexamethylene carbonate) diols. Includes system polyols and the like. These polyols can be used alone or in combination of two or more.

Examples of the above polyisocyanates include toluene diisocyanate (TDI), triphenyl-methane-4,4', 4 "-triisocyanate, benzene-1,3,5-triisocyanate, and toluene-2,4,6-triisocyanate. Isocyanate, diphenyl-2,4,4'-triisocyanate, hexamethylene diisocyanate (HDI), xylene diisocyanate, chlorophenylene diisocyanate, diphenylmethane-4,4'-diisocyanate, naphthalene-1,5-diisocyanate, xylene-α, α 'Diisocyanate, 3,3'-dimethyl-4,4'-biphenylene-diisocyanate, 3,3'-dimethoxy-4,4'-sulfonylbis (phenylisocyanate), 4,4'-methylenediol-triisocyanate, ethylene Includes diisocyanate, ethylene diisothiocianate, trimethylene diisocyanate, dicyclohexylmethane-4,4'-diisocyanate, isophorone diisocyanate, 1,6-hexamethylene diisocyanate, and 2,2,4-trimethyl-1,6-hexane diisocyanate. These polyisocyanates can be used alone or in combination of two or more.

Examples of the foaming agent include dichloromethane (methylene chloride), pentane, cyclopentane, hexane, cyclohexane and carbon dioxide in addition to water. These foaming agents may be used alone or in combination of two or more.

This step may be performed in the presence of a catalyst. Examples of the above catalysts include tertiary amines (amine catalysts) such as triethylenediamine, N, N-dimethylaminoethanol, N, N', N'-trimethylaminoethylpiperazine, tin octylate (tinoctate) and Examples thereof include organic metal compounds (metal catalysts) such as dibutyltin laurate (dibutyltin dilaurate), acetates, and alkali metal alcoholates. These catalysts can be used alone or in combination of two or more. From the viewpoint of enhancing the effect of the catalyst, the catalyst is preferably a combination of an amine catalyst and a metal catalyst.

This step may be performed together with a foam stabilizer. Examples of the defoaming agent include silicone compounds, anionic surfactants such as sodium dodecylbenzenesulfonate and sodium lauryl sulfate, polyethersiloxanes, and phenolic compounds. These foam stabilizers can be used alone or in combination of two or more.

(Step of immersing polyurethane foam in calcification solution)
As the calcification solution, the same solution as in the fourth embodiment can be used. Immersion can also be performed in the same manner as in the fourth embodiment.

(effect)
The polyurethane foam produced in this manner has a higher adhesion rate of hydroxyapatite than the conventional polyurethane foam. Therefore, hydroxyapatite can further promote bone formation between the polyurethane foam and the bone, and the polyurethane foam can be firmly bonded to the bone in a shorter period of time than before. In addition, the polyurethane foam thus produced can precipitate more hydroxyapatite than conventional polyurethane foams. Further, by imparting a chitin-derived functional group to the polyurethane foam, it is expected that the polyurethane foam has a higher adsorbing ability than the conventional adsorbent as an adsorbent for environmental pollutants.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples.

[Example 1] Production of soluble phosphorylated chitin 10 g of dry powdered chitin was dispersed in a mixed solution in which 100 mL of hexane and dimethylformamide (DMF) had a volume ratio of 50/50. After the turbidity of the dispersion medium due to dispersion was visually observed, 1 g of methanesulphonic acid was added as a catalyst while stirring the dispersion at 25 ° C., and 5 g of phosphorus pentoxide dissolved in ethanol was added at 10-minute intervals. Was added 3 times and reacted for 2 hours. Then, distilled water was added to the reaction solution, 1M NaOH was added to neutralize the pH, and then dialysis with distilled water was repeated. After dialysis was performed until there was no change in the electrical conductivity of the external solution after dialysis, the internal solution was freeze-dried.

FIG. 1 is an infrared absorption spectrum measured for a phosphorylated and solubilized chitin sample obtained from the internal solution after the above method. A peak (indicated by an arrow in the figure) characteristic of an organophosphate group was observed around 900 to 1150 cm- ^1.

FIG. 2 shows two types of chitin samples obtained by phosphorylation and solubilization obtained from the internal solution after the above method, and infrared measurements of a non-phosphorylated chitin sample without the above method. It is an absorption spectrum. As shown in FIG. 2, all the samples phosphorylated using an aprotic solvent clearly showed a peak characteristic of an organophosphate group in the vicinity of ^{900 to 1150 cm -1.} The above characteristic peak was not clearly seen in non-phosphorylated chitin.

Before and after phosphorylation, 10 mg of chitin was placed in a Kjeldahl flask, 1.5 ml of concentrated sulfuric acid and 0.5 ml of perchloric acid were added, and the mixture was heated until it became transparent. After the sample was quantitatively diluted with distilled water, the liberated phosphoric acid was measured by the molybdenum blue colorimetric method. As a result, assuming that the chitin sample before phosphorylation does not contain phosphorus and that uniform phosphorylation proceeds during phosphorylation, about 0.5% of the N-acetylglucosamine unit of chitin. It was found that the degree was phosphorylated.

From this result, it was found that chitin was phosphorylated and solubilized in water or DMF by the method according to the present invention.

[Example 2] Production of soluble phosphorylated collagen 1 g of bovine skin-derived collagen is dissolved in 300 mL of 1 M potassium phosphate buffer (pH 3), and an ice-cooled or circulating cooling device is used to avoid temperature rise (25). After adding 1 g of methanesulphonic acid as a catalyst with stirring, 1 g of phosphorus pentoxide was added 3 times at 10-minute intervals and reacted for 3 hours. After that, while cooling the reaction product, 1M NaOH was added until the pH reached 7. Then, distilled water was added to the reaction solution, and the distilled water was dialyzed to remove the reaction reagent, and then freeze-dried.

[Example 3] Phosphoration and solubilization method of insoluble collagen and gelatin The phosphorylation of collagen in which insoluble collagen is mostly composed, for example, bovine bone collagen and adult bovine skin collagen, is largely the same as the phosphorylation method of chitin. Need to be used. That is, 1 to 5 g of insoluble collagen was dispersed in a mixed solution in which 300 mL of hexane and dimethylformamide (DMF) had a volume ratio of 50/50. After adding 1 g of methanesulphonic acid as a catalyst while stirring the dispersion at 25 ° C. or lower, 5 g of phosphorus pentoxide dissolved in ethanol was added 3 times at 10-minute intervals, and the reaction was carried out for 2 hours. I let you. The dissolved component was centrifuged from the insoluble component, and distilled water was added to the reaction solution to the supernatant to neutralize the pH by adding 1M NaOH, and then dialysis was performed on the distilled water. After dialysis was performed until there was no change in the electrical conductivity of the external solution after dialysis, the internal solution was freeze-dried.

The insoluble component was solubilized and phosphorylated again using the above solvent, phosphorylation reagent, and catalyst at a temperature of 60 ° C. By this operation, most of the insoluble collagen was solubilized and could be recovered as phosphorylated gelatin. Phosphorylated gelatin was also freeze-dried after dialysis with distilled water.

FIG. 3 is an infrared absorption spectrum of two types of phosphorylated collagen samples obtained in this example and a collagen sample derived from bovine skin that has not been phosphorylated. As shown in FIG. 3, bovine-derived collagen phosphorylated by the method of the present invention is phosphorylated at a peak (indicated by an arrow in the figure) near ^{900 to 1150 cm-1, which is characteristic of an organic phosphate group.} It was more prominent than bovine collagen that was not present. The above peak was not significantly observed in bovine collagen that was not phosphorylated by the method of the present invention.

From this result, it was found that collagen was phosphorylated and solubilized in water by the method according to the present invention.

[Example 4] Separation of phosphorylated chitin bound to titanium An aqueous solution prepared by dissolving the phosphorylated chitin produced in Example 1 in water was prepared. A column (inner diameter 16 mm × height 5 cm) packed with titanium particles having a diameter of 45 μm (Osaka Titanium Technologies Co., Ltd.) was prepared. After adding 50 mL of the above aqueous solution to a column equilibrated with phosphate buffered saline (PBS) (pH 7.4), phosphate buffered saline (PBS) (pH 7.4) is added to the non-adsorbed component. Was eluted. Then, a 25 mM aqueous sodium hydroxide solution was added to elute the adsorbed component (phosphorylated chitin). FIG. 4 is a chromatogram showing the dissolution pattern in this example. The arrows in FIG. 4 indicate the timing of addition of the above aqueous solution, PBS, sodium hydroxide aqueous solution and PBS for cleaning, respectively, from the left. Non-adsorbed component to titanium (after addition of aqueous solution until immediately before addition of sodium hydroxide aqueous solution) and adsorbed component (after addition of sodium hydroxide aqueous solution, second addition of PBS) measured from the area ratio of the above chromatogram. The quantity ratio with (until just before) was 50:50.

From this result, it was found that 50% of the phosphorylated chitin produced in Example 1 had the property of binding to titanium.

[Example 5] Separation of phosphorylated collagen bound to titanium An aqueous solution prepared by dissolving the phosphorylated collagen produced in Examples 2 and 3 in water was prepared. A column (inner diameter 16 mm × height 5 cm) packed with titanium particles having a diameter of 45 μm (Osaka Titanium Technologies Co., Ltd.) was prepared. After adding 4 mg of the above aqueous solution to the column equilibrated with phosphate buffered saline (PBS) (pH 7.4), hydrochloric acid (pH 4.0) was added to elute the non-adsorbed components. Then, a 25 mM aqueous sodium hydroxide solution was added to elute the adsorbed component (phosphorylated chitin). Similarly, the same operation was performed on collagen that was not phosphorylated by the method of the present invention. FIG. 5 is a chromatogram showing the elution pattern of phosphorylated collagen. FIG. 6 is a chromatogram showing the elution pattern of collagen that has not been phosphorylated. The arrows in FIGS. 5 and 6 indicate the timing at which the aqueous solution and the sodium hydroxide aqueous solution were added, respectively. The amount ratio of the non-adsorbed component (after the addition of the aqueous solution to just before the addition of the sodium hydroxide aqueous solution) and the adsorbed component (after the addition of the sodium hydroxide aqueous solution) measured from the area ratio of the chromatogram is It was 60:40 in FIG. 5 and 85:15 in FIG.

From this result, it was found that the phosphorylated collagen produced in Examples 2 and 3 had a stronger affinity for titanium than the collagen not subjected to the phosphorylation treatment.

[Example 6] Production of composite of phosphorylated chitin and titanium and implantation in a living body A titanium non-woven fabric made of titanium fibrils having a diameter of 50 μm was cut out into a disk shape having a diameter of 2 mm and a height of 3 mm. In addition, phosphorylated chitin (adsorbed component in the chromatography of Example 4) was dissolved in PBS to prepare a 0.1% phosphorylated chitin solution. After immersing the molded body of the titanium non-woven fabric in the phosphorylated chitin solution for a certain period of time, it is dried, and the surface of each titanium fibril is coated with phosphorylated chitin. A complex with and was produced. On the other hand, as a biological implant of a comparative example, a titanium non-woven fabric molded body not coated with phosphorylated chitin was also prepared.

Eight-week-old Wister rats were anesthetized by intraperitoneal administration of 8% chloral hydrate. A 2.8 mm diameter hole was drilled in the tibia using a dental drill and implants of Examples and Comparative Examples were implanted. Two and six weeks after implantation, the implant and its surroundings were removed. Sections of the obtained samples were prepared, the bone tissue was stained with the Villanueva Osteochrome staining method, and the amount of bone formed around the implant was compared.

7A and 7B are tissue specimens from rats with implants of Examples. FIG. 7B is a partial enlargement of FIG. 7A. 7C and 7D are tissue specimens from rats implanted with comparative implants. FIG. 7D is a partial enlargement of FIG. 7C. In FIGS. 7A and 7B, a vigorous proliferation (dark colored part) of the substrate of the stained bone tissue and the bone cells was observed. In FIGS. 7C and 7D, although there was some cell aggregation, the vigorous bone formation as in FIGS. 7A and 7B was not observed as a whole. Comparing the areas of the new bones in the titanium non-woven fabric, there was a 4.4-fold difference between the examples and the comparative examples.

From this result, it was found that the complex of phosphorylated chitin and titanium produced by the method of the present invention promotes bone formation when implanted in the body. Therefore, it is considered that this complex can further promote bone formation between the titanium base material and the bone, and can bond the titanium base material to the bone in a shorter period of time and more firmly.

[Example 7] Precipitation of hydroxyapatite on the surface of a complex of phosphorylated chitin and titanium 15 mM / L calcium chloride (CaCl ₂ ), 9 mM / L dipotassium phosphate (K ₂ HPO4), 0.7 M A calcification solution containing / L sodium chloride (NaCl) and 0.02 M / L sodium hydrogen carbonate (NaHCO _{3) was prepared.} The complex of phosphorylated chitin and titanium obtained in Example 6 was immersed in the calcification solution whose pH was adjusted to 6.01 by bubbling carbon dioxide gas, and incubated at 37 ° C. for 48 hours. Then, the titanium base material was taken out, washed with desalted water, and freeze-dried. The lyophilized titanium substrate was reimmersed in the new calcification solution and incubated every other day for 2 weeks, changing the calcification solution. Then, the titanium base material was taken out, washed with desalted water, and freeze-dried. The titanium substrate on which the hydroxyapatite was precipitated thus obtained was stored in a sterile container in a dry state at 10 ° C. or lower.

8A and 8B are scanning electron microscope (SEM) photographs of the surface of the complex before the above treatment. FIG. 8B is a partial enlargement of FIG. 8A. 8C and 8D are SEM photographs of the surface of the titanium substrate on which hydroxyapatite was precipitated by the above treatment. FIG. 8D is a partial enlargement of FIG. 8C.

From this result, it was found that hydroxyapatite was uniformly precipitated on the surface of the composite of phosphorylated chitin and titanium produced by the method of the present invention. Therefore, it is considered that this complex can further promote bone formation between the titanium base material and the bone by hydroxyapatite, and can bond the titanium base material to the bone in a shorter period of time and more firmly. ..

[Example 8-1] Production of polyurethane foam (one-shot method)
A polyurethane foam was produced using the following components.

(Polyprides)
Polyether polyol, Mw3000, hydroxyl value 56 mgKOH / g, number of functional groups 3, product number: Sanniks GP-3050NS, Sanyo Chemical Industries, Ltd. ("Sanniks" is a registered trademark of the company)
(Isocyanates)
A mixture of toluene diisocyanate (TDI), 2-4TDI / 2-6TDI. The ratio is 2-4TDI / 2-6TDI = 80/20, product number: Cosmonate T-80, Mitsui Chemicals, Inc. ("Cosmonate" is a registered trademark of the company)
(Phosphorylated chitin)
The one manufactured in Example 1 is used.
(catalyst)
Amine catalyst, product number: DABCO 33-LV, Air Products Japan Co., Ltd. ("DABCO" is a registered trademark of the company)
Metal (tin) catalyst, product number: MRH-110, Johoku Chemical Co., Ltd. (foaming agent)
Silicone defoaming agent, silicone defoaming agent for soft materials, product number: SZ-1136, Toray Dow Corning Co., Ltd.

(Preparation of mixed solution)
To 100 parts by mass of the above polyols, 1 part by mass of the above phosphorylated chitin was added and stirred to prepare a mixed solution 1 in which phosphorylated chitin was dispersed in the polyols. The mixed solution 2 and the mixed solution 3 were prepared in the same manner except that the amount of the phosphorylated chitin was changed to 3 parts by mass and 5 parts by mass, respectively.

(Manufacturing of polyurethane foam)
In 101 parts by mass of the above mixed solution 1, 4.4 parts by mass of water (foaming agent), 1.0 part by mass of amine catalyst, 1.5 parts by mass of foam stabilizer and 0.3 parts by mass of metal (tin). ) A catalyst was added and stirred, and 52.8 parts by mass of isocyanate was further added and mixed and stirred to produce a polyurethane foam 1. The polyurethane foam 2 and the polyurethane foam 3 were produced in the same manner except that the mixed solution 1 was changed to 103 parts by mass of the mixed solution 2 and 105 parts by mass of the mixed solution 3. The polyurethane foam 4 was produced in the same manner except that the mixed solution 1 was changed to the above-mentioned polyols to which phosphorylated chitin was not added.

Table 1 shows the materials used for producing the polyurethane foams 1 to 4 and parts by mass thereof, and the content of phosphorylated chitin in the polyurethane foams 1 to 4. The content of the phosphorylated chitin is a value obtained by dividing the mass of the phosphorylated chitin used as a material by the mass of all the materials.

[Example 8-2] Production of polyurethane foam (prepolymer method)
A polyurethane foam was produced using the following components.

(Polyprides)
Polyethylene glycol, product number: PEG # 1000, Lion Specialty Chemicals Co., Ltd. (isocyanates)
A mixture of toluene diisocyanate (TDI), 2-4TDI / 2-6TDI. The ratio is 2-4TDI / 2-6TDI = 80/20, product number: Cosmonate T-80, Mitsui Chemicals, Inc. (foaming agent)
Purified water (foaming agent)
ADEKA PLRONIC L61, manufactured by ADEKA CORPORATION
(Other)
Purified glycerin, manufactured by Kao Corporation

(Manufacturing of prepolymer)
The initiator is ethylene glycol, 61.69 parts by mass of the above-mentioned polyol and 3.32 parts by mass of glycerin are mixed, and 35 parts by mass of the above-mentioned isocyanates are placed in a reaction vessel and mixed and reacted at 100 ° C. for 4 hours. , A urethane prepolymer having a reactive isocyanate group content of 8.58% by mass was obtained.

(Preparation of mixed solution)
To 100 parts by mass of the foaming agent, 5 parts by mass of the phosphorylated chitin and 1 part by mass of the foam stabilizer were added and stirred to prepare a mixed solution 4 in which the phosphorylated chitin was dispersed in water. The mixed solution 5 was prepared in the same manner except that the amount of the phosphorylated chitin was changed to 10 parts by mass.

(Manufacturing of polyurethane foam)
106 parts by mass of the mixed solution 4 was mixed and stirred with respect to 100 parts by mass of the urethane prepolymer at a liquid temperature of 25 ° C., and then poured into a foam container to obtain a foam. The obtained foam was dried by microwave at 1.5 kW for 10 minutes to produce a polyurethane foam 5. The polyurethane foam 6 was produced in the same manner except that the mixed solution 4 was changed to 111 parts by mass of the mixed solution 5. The polyurethane foam 7 was produced in the same manner except that the mixed solution 4 was changed to a mixed solution of a foaming agent to which phosphorylated chitin was not added and the above-mentioned surfactant.

Table 2 shows the materials used for producing the polyurethane foams 5 to 7, parts by mass thereof, and the content of phosphorylated chitin in the polyurethane foams 5 to 7. Since the purified water as the foaming agent is removed by drying, the content of the phosphorylated chitin is the mass of the phosphorylated chitin used as the material, and all the materials except the purified water (in the table, It is a value obtained by dividing by the mass of (expressed as "solid content").

[Example 9] Polyurethane foam calcification Test piece 1 is a polyurethane foam 4 containing no phosphorylated chitin in Example 8-1, and polyurethane foam 4 is coated with phosphorylated chitin according to the method of Example 6. The coated polyurethane foam 4'was used as test piece 2, and the polyurethane foam 2 containing 1.84% phosphorylated chitin in Example 8-1 was used as test piece 3. Hydroxyapatite was precipitated on each test piece by the same method as in Example 7.

FIG. 9 shows the average mass of each polyurethane foam before precipitating hydroxyapatite (referred to as “original sample” in the figure), and test piece 1 and test piece after precipitating hydroxyapatite, respectively. It is a graph which shows the mass of 2 and the test piece 3. The mass of the test piece 1 was increased by 1.5 times, the mass of the test piece 2 was increased by 1.7 times, and the mass of the test piece 3 was increased by 2.3 times with respect to the above average mass (7.6 mg). ..

From this result, the polyurethane foam coated with phosphorylated chitin precipitates more hydroxyapatite than the polyurethane foam not coated with phosphorylated chitin, and the polyurethane foam produced by mixing phosphorylated chitin is It was found that more hydroxyapatite was precipitated.

Chitin and collagen are biopolymers that are abundant in nature. If these biopolymers are solubilized by phosphorylation, it is possible to provide a new basic material for creating a biologically functional material. In addition, phosphorylated chitin and collagen are used for medical applications through the formation of a complex with titanium and the preparation of functional polyurethane, the production of a large-scale 3D cell culture base for regenerative medicine, an adsorbent for environmental purification, and immunotherapy. It has potential in a wide range of fields such as the production of mass cell culture bases for, and the preparation of novel functional nanofibers by hybridizing with nanocell rose fibers.

Claims (4)

The process of dispersing chitin in an aprotic solvent,
A step of phosphorylating the dispersed chitin in the presence of a catalyst,
A step of separating phosphorylated chitin that binds or adsorbs to titanium and phosphorylated chitin that does not bind or adsorb to titanium by affinity chromatography using titanium beads as a stationary phase from the phosphorylated chitin.
A solution containing the phosphorylated chitin bound or adsorbed to the separated titanium, a step of binding or adsorbing the titanium substrate, the phosphorylated chitin by contacting the titanium substrate,
including,
A method for producing a complex of phosphorylated chitin and titanium. The method for producing a composite of phosphorylated chitin and titanium according to claim 1, further comprising a step of immersing a titanium base material in contact with the solution containing phosphorylated chitin in a calcification solution. Possess a base material made of titanium or a titanium alloy, and a phosphorylated chitin-containing layer covering at least a portion of a surface of said substrate, said phosphorylated chitin-containing layer, as a phosphorylated chitin, bound or adsorbed to a titanium A complex that is a layer in which only phosphorylated chitin to be bonded or adsorbed on the surface of the base material. The complex according to claim 3, wherein the phosphorylated chitin-containing layer contains hydroxyapatite.

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