CN117045861A - Titanium-mineral collagen hydrogel suitable for bone repair material and preparation method thereof - Google Patents

Abstract

The application discloses a titanium-mineral collagen hydrogel suitable for a bone repair material and a preparation method thereof. The application takes the natural collagen which is the main structural protein of cartilage as a template, takes the natural macromolecular carboxymethyl chitosan as an inducer, takes the titanyl sulfate solution as a precursor solution, and drives the hydrolysis polycondensation of the titanyl sulfate to form titanium dioxide (TiO based on the principle of biomimetic mineralization ₂ ) Deposited on the collagen fiber, and the collagen fiber generate strong interface interaction to prepare the high-strength titanium-treated collagen hydrogel. The prepared mineralized hydrogel improves the mechanical property of pure collagen hydrogel and simultaneously has the advantages of good bone conduction capacity, biocompatibility, hemostasis and bacteriostasis. The method comprisesThe hydrogel can be used as a bone repair material alone or in combination with other biological factors and drugs.

Description

A titanium mineralized collagen hydrogel suitable for bone repair materials and preparation method thereof

Technical field

The invention belongs to the technical field of collagen-based medical materials and relates to a titanium mineralized collagen hydrogel suitable for bone repair materials and a preparation method thereof.

Background technique

Bone tissue damage is one of the serious health problems worldwide. In current clinical practice, bone transplantation is the most common bone repair method, mainly including autologous bone, allogeneic bone and tissue-engineered artificial bone transplantation. However, autologous bone transplantation has the disadvantages of limited bone supply sources and donor site complications; homologous bone transplantation has the disadvantages of limited bone supply and donor site complications. Allogeneic bone transplantation has limited osteoinductivity, immunogenicity and immune response. These limitations make the development of bone repair materials in bone tissue engineering a current research hotspot, providing a new treatment strategy and ideal scaffold for bone injuries. Materials should have good biocompatibility, non-toxicity, low cost, non-carcinogenicity and excellent bone conduction properties. Hydrogel has a three-dimensional network structure similar to that of extracellular matrix. The mild environment can protect cells and facilitate the even distribution of cells in three-dimensional space, provide structural support for defective parts, and enable bone defects to be repaired through intrinsic healing mechanisms; more importantly What’s more, by utilizing the transformation characteristics from solution to gel, injection implantation can be achieved when repairing tissues with complex geometric shapes, and surgical trauma can be reduced through in-situ molding.

Collagen is the main structural protein that constitutes cartilage and bone. It is an essential nutrient for bone tissue metabolism. It can provide energy and raw materials for bone repair and is a potential candidate for the development of cartilage tissue materials. Compared with collagen molecules, the materials constructed by collagen fibers are very similar to the microstructure of collagen in tissues. They have the advantages of slow degradation, high integration with surrounding tissues, and suppression of inflammation, and can perform their biological functions more effectively. Collagen hydrogel formed by coating an aqueous solution with collagen fibers is a natural hydrogel with high water content, fast swelling, stable structure and good biocompatibility. It can be used as a bionic scaffold in cartilage and bone tissue engineering. All aspects have broad research and development prospects. However, the mechanical strength of pure collagen hydrogel is not high and cannot be directly used in tissue engineering. Therefore, modifying collagen hydrogel to improve its mechanical strength is a current research hotspot.

At present, there are three main methods to improve the strength of collagen hydrogel: 1) Physical method: no exogenous substances are introduced, which avoids the entry of exogenous toxic and harmful chemicals into the collagen hydrogel, but it is difficult to obtain a uniform cross-linking method. co-product; 2) Chemical method: its thermal stability and mechanical properties are improved, but its biodegradability and biocompatibility are poor; 3) Blending modification: Although the excellent properties of the two blend materials are retained performance, but its mechanical strength is not significantly improved.

Biomineralization is a process in which biological macromolecules and some induction factors regulate the orderly nucleation and growth of inorganic minerals under a certain physiological environment to produce biological minerals with fine structures. The biomineralized material formed by this process is an organic-inorganic nanohybrid material in nature. It has a multi-level structural organization from nanometers, micrometers to macroscopic scales, and has high hardness and fracture resistance. For example, hard tissues such as human bones and teeth. Bone tissue is a complex biomineralized material, mainly composed of collagen, hydroxyapatite and non-collagen proteins through biomineralization. Collagen acts as a bone scaffold to give it elasticity and toughness, while hydroxyapatite and collagen fibers present organic -Identification and fitting of inorganic interfaces to provide stiffness. In addition, the multiple fibril structure of collagen fibers lays the foundation for the multi-level micro-nano structure of bone tissue. This strict hierarchical structure provides bone tissue with superior mechanical properties. Inspired by this, the use of bionic mineralization technology to combine hydroxyapatite or other inorganic substances with collagen to prepare bone repair materials has become a research hotspot. However, the materials prepared are mostly membrane-like or sponge-like, and this technology is rarely used to prepare high-quality bone repair materials. Strong collagen hydrogel and retains the fiber structure of collagen in the hydrogel.

Contents of the invention

In view of the problems existing in the prior art, the present invention provides a titanium mineralized collagen hydrogel suitable for bone repair materials and a preparation method thereof, thereby solving the technical problem of poor mechanical properties of collagen hydrogel in the prior art.

The present invention is realized through the following technical solutions:

A method for preparing titanium mineralized collagen hydrogel suitable for bone repair materials, including the following steps:

S1: Dissolve collagen sponge and carboxymethyl chitosan in sodium salt solution respectively to prepare collagen solution and carboxymethyl chitosan solution;

S2: Add the carboxymethyl chitosan solution dropwise into the collagen solution, stir and react, and obtain a collagen-carboxymethyl chitosan composite solution;

S3: Make the collagen-carboxymethyl chitosan composite solution self-assemble into fibers to prepare the collagen-carboxymethyl chitosan composite hydrogel;

S4: Place the collagen-carboxymethyl chitosan composite hydrogel in a zirconium cross-linked solution to react to prepare a zirconium cross-linked collagen-carboxymethyl chitosan composite hydrogel;

S5: Place the zirconium cross-linked collagen-carboxymethyl chitosan composite hydrogel in a titanyl sulfate solution to perform a mineralization reaction to prepare the titanium mineralized collagen hydrogel suitable for bone repair materials. .

Preferably, in step S1, the salt concentration in the sodium salt solution is 100-200 mmol/L; the mass concentration of the collagen solution is 0.4%-2%.

Preferably, in step S2, the mass ratio of collagen to carboxymethyl chitosan in the collagen-carboxymethyl chitosan composite solution is 1: (0.25-4); the pH value of the reaction system in step S2 is controlled as 7～8, reaction temperature is 2～10℃.

Preferably, in step S3, the collagen-carboxymethyl chitosan composite solution is placed at 30-37°C for self-assembly to prepare the collagen-carboxymethyl chitosan composite hydrogel.

Preferably, in step S4, the zirconium cross-linked solution is prepared by dissolving trisodium citrate and zirconium sulfate tetrahydrate in water, wherein the mass ratio of trisodium citrate and zirconium sulfate tetrahydrate is 1:( 1 to 5); the mass concentration of zirconium sulfate tetrahydrate in the zirconium cross-linking solution is 0.1% to 5%.

Preferably, in step S4, the volume ratio of the collagen-carboxymethyl chitosan composite hydrogel and zirconium cross-linking solution is 1:(1~10); the reaction temperature in step S4 is 25~40°C, The reaction time is 48~96h.

Preferably, in step S5, the mass concentration of the titanyl sulfate solution is 0.1% to 20%; the volume ratio of the zirconium cross-linked collagen-carboxymethyl chitosan composite hydrogel to the titanyl sulfate solution is 1:(1～10).

Preferably, in step S5, the mineralization process is specifically: after mixing the zirconium cross-linked collagen-carboxymethyl chitosan composite hydrogel and the titanyl sulfate solution, the mixture is then placed at 25-40°C. Mineralization is carried out for 24 to 72 hours to prepare the titanium mineralized collagen hydrogel suitable for bone repair materials.

A titanium mineralized collagen hydrogel suitable for bone repair material is prepared by the above method. The storage modulus of the titanium mineralized collagen hydrogel used for bone repair is 17.8 to 264.5 kPa.

A bone repair material includes the above-mentioned titanium mineralized collagen hydrogel suitable for bone repair materials.

Compared with the existing technology, the present invention has the following beneficial technical effects:

A titanium mineralized collagen hydrogel suitable for bone repair materials and a preparation method thereof, using collagen fibers that self-assemble into a multi-layered structure as a template, natural macromolecule carboxymethyl chitosan as an inducer, and titanyl sulfate solution is the precursor solution. Based on the principle of bionic mineralization, titanyl sulfate is driven to undergo hydrolysis and condensation mineralization inside and on the fiber to form a composite hydrogel of TiO ₂ and collagen fibers. This process creates a strong interface interaction between inorganic matter and collagen fibers. function, thus giving the collagen hydrogel better mechanical properties; TiO ₂ has good biocompatibility, osteoconductivity and excellent mechanical properties, can induce the deposition of hydroxyapatite, and is an active ingredient for osseointegration in the body. Endow the hydrogel with good bone conduction and bone repair functions. In addition, carboxymethyl chitosan is an amphoteric polyelectrolyte that can not only induce the hydrolysis and condensation of titanyl sulfate, but also give the composite hydrogel good hemostatic and antibacterial properties, and improve the material's ability to promote the regeneration and repair of articular cartilage. . At the same time, during the preparation process, zirconium cross-linking solution was used to protect the collagen-carboxymethyl chitosan composite hydrogel, and then it was placed in titanyl sulfate solution for titanium mineralization, which effectively prevented the collagen fibers from being damaged under strong acid conditions. is destroyed, effectively maintaining the multi-layered fiber structure of the hydrogel material.

Further, the salt concentration in the sodium salt solution is 100-200mmol/L, and sodium ions and other anions perform charge shielding on collagen and carboxymethyl chitosan, preventing agglomeration after mixing collagen and carboxymethyl chitosan; The mass concentration of the collagen solution is 0.4% to 2%. If the concentration is too low, the fibers formed by self-assembly will be immature. If the concentration is too high, the viscosity of the system will be too high, making it difficult to form a uniform system with carboxymethyl chitosan. , this concentration range can ensure the formation of a good fiber structure without making the system viscosity too high, effectively meeting experimental needs.

Furthermore, the mass ratio of collagen to carboxymethyl chitosan in the collagen-carboxymethyl chitosan composite solution is 1: (0.25~4). If the ratio is too high or too low, the compatibility of the blend solution will be poor. Poor, it is not conducive to producing products with good performance. Control the pH value of the reaction system in step S2 to 7 to 8. This pH value can effectively realize the subsequent self-assembly process, and at this pH value, the amount of electrostatic charge carried by the collagen is the least, higher than this range or lower than this range. range, the charge on the collagen surface is too high, and the electrostatic force between the collagen and carboxymethyl chitosan is too strong, and the two are prone to agglomeration. The reaction temperature of the reaction system in step S2 is controlled to be 2-10°C. This temperature can effectively maintain the triple helix structure of collagen, that is, its biological activity, and prevent self-assembly of collagen when blended with carboxymethyl chitosan.

Further, in step S3, specifically, the collagen-carboxymethyl chitosan composite solution is placed at 30-37°C for self-assembly to prepare the collagen-carboxymethyl chitosan composite hydrogel. Effectively realizes the self-assembly process of collagen.

Further, in step S4, the zirconium cross-linked solution is prepared by dissolving trisodium citrate and zirconium sulfate tetrahydrate in water. Zirconium cross-linking effectively improves the strength of the collagen-carboxymethyl chitosan composite hydrogel. Acid resistance, because the titanyl sulfate solution has strong acidity during the subsequent mineralization process. If the collagen-carboxymethyl chitosan composite hydrogel is directly placed in the titanyl sulfate solution, the fibers assembled from collagen molecules will The structure will be destroyed, the advantages of collagen fibers will be lost, and the hydrogel will even be dissociated into a solution and modified hydrogel materials cannot be produced. Trisodium citrate mainly coordinates with zirconium sulfate. After adding it, it can increase the pH value of the zirconium sulfate solution and avoid the strong acidity of pure zirconium sulfate solution from damaging collagen fibers; trisodium citrate and zirconium sulfate tetrahydrate The mass ratio of the substances is 1: (1 ~ 5); the mass concentration of zirconium sulfate tetrahydrate in the zirconium cross-linking solution is 0.1% ~ 5%. The zirconium cross-linking process mainly affects the collagen-carboxymethyl chitosan composite The fiber structure in the hydrogel plays an effective protective role; when the concentration of zirconium sulfate tetrahydrate is too low, the purpose of protection and fixation cannot be achieved, and the concentration of zirconium sulfate solution is too high, the system is too acidic, and a large amount of water needs to be added. of trisodium citrate, resulting in a waste of raw materials and an increase in impurities in the system.

Further, in step S4, the volume ratio of the collagen-carboxymethyl chitosan composite hydrogel to the zirconium cross-linking solution is 1: (1-10), so that the zirconium cross-linking effect on the collagen-carboxymethyl chitosan The sugar complex hydrogel forms an effective protection to avoid its solubilization during the later mineralization process. In addition, the reaction temperature in step S4 is 25 to 40°C and the reaction time is 48 to 96 hours. These reaction parameters effectively improve the cross-linking efficiency of zirconium and collagen-carboxymethyl chitosan composite hydrogel, thereby improving the water quality. Acid resistance of the gel.

Further, in step S5, the mass concentration of the titanyl sulfate solution is 0.1% to 20%; the volume ratio of the zirconium cross-linked collagen-carboxymethyl chitosan composite hydrogel to the titanyl sulfate solution is 1: (1-10), achieving full functionalization of the interior and surface of the collagen-carboxymethyl chitosan composite hydrogel by TiO ₂ .

Description of the drawings

In order to explain the technical solutions of the embodiments of the present invention more clearly, the drawings required to be used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and therefore do not It should be regarded as a limitation of the scope. For those of ordinary skill in the art, other relevant drawings can be obtained based on these drawings without exerting creative efforts.

Figure 1 is a schematic flow chart of a method for preparing titanium mineralized collagen hydrogel suitable for bone repair materials in the present invention;

Figure 2 is a scanning electron microscope (SEM) image of the collagen hydrogel prepared in Comparative Example 1 and the titanium mineralized collagen hydrogel prepared by the technical solution of the present invention; wherein, a-collagen hydrogel, b-High strength titanium mineralized collagen hydrogel.

Figure 3 shows the collagen hydrogel prepared in Comparative Example 1 and the titanium mineralized collagen hydrogel prepared in Example 1 of the present invention, that is, the storage medium of the titanium mineralized collagen hydrogel suitable for bone repair materials in the present invention. Energy modulus comparison chart, where a-collagen hydrogel, b-high-strength titanium mineralized collagen hydrogel.

Detailed ways

In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the terms and expressions mentioned in the description and claims are generally described and defined below. Unless otherwise specified, all technical and scientific terms used in the text have their usual meanings as understood by those skilled in the art regarding the present invention. In the event of conflict, the definitions in this specification shall prevail.

The theories or mechanisms described and disclosed herein, whether true or false, should not limit the scope of the invention in any way, that is, the invention may be implemented without being limited to any particular theory or mechanism.

In this article, all characteristics such as numerical values, quantities, contents, and concentrations defined in the form of numerical ranges or percentage ranges are for simplicity and convenience only. Accordingly, a description of a numerical range or percentage range shall be deemed to cover and specifically disclose all possible subranges and individual values within the range (including integers and fractions).

In this article, unless otherwise stated, "comprises", "includes", "contains", "has" or similar terms cover the meanings of "consisting of" and "consisting essentially of", for example, "A includes a ” covers the meaning of “A contains a and others” and “A contains only a”.

Herein, in order to keep the description concise, not all possible combinations of each technical feature in each embodiment or example are described. Therefore, as long as there is no contradiction in the combination of these technical features, each technical feature in each embodiment or example can be combined in any way, and all possible combinations should be considered to be within the scope of this specification.

As shown in Figure 1, the present invention provides a titanium mineralized collagen hydrogel suitable for bone repair materials and a preparation method thereof, specifically:

S1: Dissolve collagen sponge and carboxymethyl chitosan in sodium salt solution respectively to prepare a collagen solution with a mass concentration of 0.4% to 2% and a carboxymethyl chitosan solution with a mass concentration of 0.1% to 8%. , control the pH value of each solution during the dissolution process to be 7 to 8, and the temperature to be 2 to 10°C;

Wherein, the salt concentration in the sodium salt solution is 100-200mmol/L, more preferably 120-160mmol/L. The sodium salt is to shield the charges of collagen and carboxymethyl chitosan and prevent collagen and carboxymethyl chitosan from being charged. Precipitation occurs after mixing; the mass concentration of the collagen solution is 0.4% to 2%, more preferably 0.6% to 1.6%. If the concentration of collagen is too low, the fibers formed by self-assembly will be immature, and if the concentration is too high, it will cause If the viscosity of the system is too high, it is difficult to form a uniform system with carboxymethyl chitosan. This concentration range can ensure the formation of a good fiber structure without making the system viscosity too high, effectively meeting the experimental needs. The mass concentration of the carboxymethyl chitosan solution is 0.1% to 8%, more preferably 0.3% to 3.2%.

In addition, the sodium salt solution is selected from at least one of disodium hydrogen phosphate, sodium dihydrogen phosphate or sodium chloride. The reason why sodium salt is chosen here is that firstly, collagen is insoluble in deionized water, and secondly, sodium salt can shield the charges of collagen and carboxymethyl chitosan to prevent their precipitation. In addition, collagen self-assembly requires the participation of salt.

S2: Slowly drop the carboxymethyl chitosan solution into the collagen solution, stir and react, and obtain a collagen-carboxymethyl chitosan composite solution, in which there is a gap between collagen and carboxymethyl chitosan. Bonded through electrostatic and hydrogen bonding.

Among them, the dry weight mass ratio of collagen to carboxymethyl chitosan is 1:(0.25~4), that is, the mass ratio of collagen to carboxymethyl chitosan in the collagen-carboxymethyl chitosan composite solution is 1:( 0.25~4), more preferably 1:(0.5~2). If the ratio is too high or too low, the blend solution will have poor compatibility, which is not conducive to the production of products with good performance; in addition, carboxymethyl chitosan The amount is related to the mineralization effect, because the subsequent mineralization process of titanyl sulfate mainly relies on the induction effect of carboxymethyl chitosan. It was found in the experiment that when carboxymethyl chitosan is not added, the hydrolysis and condensation polymerization process of titanyl sulfate Very slowly, resulting in mineralization taking too long and too little titanium dioxide being deposited. Control the pH value of the reaction system in this step to 7 to 8, more preferably 7.2 to 7.6. This pH value can effectively realize the self-assembly process. At this pH value, the charge on the collagen surface is the smallest, higher than this range or lower than In this range, the charge on the collagen surface is too high, and the electrostatic force between it and carboxymethyl chitosan is too strong, easily causing precipitation. Controlling the reaction temperature at 2 to 10°C can effectively maintain the stability of collagen and prevent self-assembly when blended with carboxymethyl chitosan, and control the stirring reaction for 12 to 24 hours, more preferably 16 to 20 hours, so that collagen The combination with carboxymethyl chitosan is more complete.

S3: Place the collagen-carboxymethyl chitosan composite solution for self-assembly at 30-37°C for 3 hours to prepare the collagen-carboxymethyl chitosan composite hydrogel. During this process, the collagen molecules are assembled into fibers. , this temperature effectively realizes the self-assembly process of collagen and the formed collagen fibers are relatively mature.

S4: Mix the collagen-carboxymethyl chitosan composite hydrogel with the zirconium cross-linked liquid, stir and react, and prepare a zirconium cross-linked collagen-carboxymethyl chitosan composite hydrogel; the reason why here The collagen-carboxymethyl chitosan composite hydrogel needs to be cross-linked with zirconium, mainly because the titanyl sulfate solution is highly acidic. If the collagen-carboxymethyl chitosan composite hydrogel is directly placed in sulfuric acid In the oxygen-titanium solution, the collagen fiber structure will be destroyed, losing the advantages of collagen fibers, and even the hydrogel will be dissociated into a solution and modified hydrogel materials cannot be produced. Therefore, the zirconium cross-linking process here plays an effective protective role on the collagen-carboxymethyl chitosan composite hydrogel.

The zirconium cross-linking solution is prepared by dissolving trisodium citrate and zirconium sulfate tetrahydrate in deionized water at room temperature, wherein the mass ratio of trisodium citrate and zirconium sulfate tetrahydrate is 1:(1~ 5), more preferably 1: (2-3.5), here, trisodium citrate mainly plays a coordination role for zirconium sulfate. After adding it, it can increase the pH value of the zirconium sulfate solution and avoid damage to the fiber. The mass concentration of zirconium sulfate tetrahydrate in the zirconium cross-linking solution is 0.1% to 5%, more preferably 0.5% to 2.5%. The zirconium cross-linking process mainly plays an effective role in the collagen-carboxymethyl chitosan composite hydrogel. Protective effect; when the concentration of zirconium sulfate tetrahydrate is too low, the purpose of protection and fixation cannot be achieved, and the concentration of zirconium sulfate solution is too high, the system is too acidic, and a large amount of trisodium citrate needs to be added, resulting in waste of raw materials. Impurities in the system increase.

The volume ratio of the collagen-carboxymethyl chitosan composite hydrogel to the zirconium cross-linking solution is 1:(1-10); the reaction temperature in step S4 is 25-40°C, more preferably 30-37°C. The reaction time is 48 to 96 hours, more preferably 60 to 80 hours. This reaction parameter effectively improves the cross-linking effect between zirconium and collagen-carboxymethyl chitosan composite hydrogel, thereby improving the acid resistance of the hydrogel.

S5: Mix the zirconium cross-linked collagen-carboxymethyl chitosan composite hydrogel with the titanyl sulfate solution, stir the reaction and perform mineralization to prepare the titanium mineralized collagen-carboxymethyl chitosan. Composite hydrogel; the mineralization process is a process in which titanyl sulfate is converted into TiO ₂ .

Wherein, the preparation process of the titanyl sulfate solution is specifically to dissolve titanyl sulfate in deionized water at room temperature to prepare a titanyl sulfate solution with a mass concentration of 0.1% to 20%, and more preferably the quality of the titanyl sulfate solution. The concentration is 1% to 10%; the volume ratio of the zirconium cross-linked collagen-carboxymethyl chitosan composite hydrogel and titanyl sulfate liquid is 1:(1-10).

The specific mineralization process is as follows: After mixing the zirconium cross-linked collagen-carboxymethyl chitosan composite hydrogel and titanyl sulfate solution, the mixture is then placed at 25-40°C for mineralization for 24-72 hours to prepare The titanium mineralized collagen-carboxymethyl chitosan composite hydrogel is obtained. Here, the mineralization temperature is more preferably 30 to 37°C, and the mineralization time is more preferably 48 to 60 hours.

The collagen sponge in this application is prepared by the following method: fresh cowhide is pre-treated with hair removal, degreasing, etc., then chopped into pieces, and then placed in a 0.5 mol/L acetic acid solution containing 3% pepsin (EC 1:10000). extract. Use a high-speed refrigerated centrifuge to centrifuge for 10 minutes to separate the supernatant. The precipitate obtained after sodium chloride salting out and centrifugation was dissolved again in 0.5 mol/L acetic acid solution. The dissolved collagen was dialyzed with 0.1 mol/L acetic acid solution for three days. The dialysate was changed every 12 hours. Finally, The collagen solution is freeze-dried, and the collagen sponge obtained after freeze-drying is stored in a desiccator for later use.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the invention and are not intended to limit the scope of the invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

In the following examples, conventional equipment in this field was used. Experimental methods without specifying specific conditions in the following examples usually follow conventional conditions or conditions recommended by the manufacturer. Various raw materials are used in the following examples. Unless otherwise stated, conventional commercially available products are used, and their specifications are conventional specifications in this field. In the description of the present invention and the following examples, unless otherwise specified, "%" means weight percentage, "part" means weight part, and proportion means weight ratio.

Comparative example 1

The difference from Example 1 is that a collagen solution with a concentration of 0.5% is directly self-assembled to prepare a collagen hydrogel.

Example 1:

Dissolve collagen sponge and carboxymethyl chitosan in sodium salt solution of 10mmol/L disodium hydrogen phosphate/sodium dihydrogen phosphate and 125mmol/L sodium chloride respectively to prepare collagen solution and carboxymethyl chitosan solution. ; The mass concentration of the collagen solution is 1%, the mass concentration of the carboxymethyl chitosan solution is 1.5%, the volumes of the collagen solution and the carboxymethyl chitosan solution are the same, and the pH value of each solution during the dissolution process is controlled to 7.2. The temperature is 4℃. Slowly add the carboxymethyl chitosan solution dropwise to the stirring collagen solution at 4°C, and stir for 16 hours to prepare a collagen-carboxymethyl chitosan composite solution. Subsequently, the composite solution was placed at 37°C for self-assembly for 3 hours to prepare a collagen-carboxymethyl chitosan composite hydrogel. Dissolve trisodium citrate and zirconium sulfate tetrahydrate in deionized water, and prepare a zirconium cross-linking solution with a mass concentration of zirconium sulfate tetrahydrate of 3%, in which the mass ratio of trisodium citrate to zirconium sulfate tetrahydrate is 1:1. The composite hydrogel was placed in the zirconium cross-linking solution and fixed with zirconium at 35°C for 72 hours. The volume ratio of the collagen-carboxymethyl chitosan composite hydrogel to the zirconium cross-linking solution was 1:5; finally, the zirconium was The fixed composite hydrogel was placed in 3% titanium sulfate solution and mineralized at 37°C for 48 hours to obtain high-strength titanium mineralized collagen hydrogel, zirconium cross-linked collagen-carboxymethyl chitosan composite hydrogel. The volume ratio of glue to titanyl sulfate solution is 1:6.

The storage modulus of the titanium mineralized collagen hydrogel suitable for bone repair materials prepared in this embodiment is 120.0 kPa.

Example 2:

Dissolve collagen sponge and carboxymethyl chitosan in 150mmol/L sodium chloride solution respectively to prepare collagen solution and carboxymethyl chitosan solution; the mass concentration of collagen solution is 2%, and carboxymethyl chitosan The mass concentration of the solution is 8%. The volumes of the collagen solution and the carboxymethyl chitosan solution are the same. The pH value of each solution during the dissolution process is controlled to be 8 and the temperature is 10°C. Slowly add the carboxymethyl chitosan solution dropwise to the stirring collagen solution at 10°C, and stir for 24 hours to prepare a collagen-carboxymethyl chitosan composite solution. Subsequently, the composite solution was placed at 37°C for self-assembly for 3 hours to prepare a collagen-carboxymethyl chitosan composite hydrogel. Dissolve trisodium citrate and zirconium sulfate tetrahydrate in deionized water, and prepare a zirconium cross-linking solution with a mass concentration of zirconium sulfate tetrahydrate of 5%, in which the mass ratio of trisodium citrate to zirconium sulfate tetrahydrate is: 1:10. The composite hydrogel was placed in the zirconium cross-linking solution and fixed with zirconium at 40°C for 96 hours. The volume ratio of the collagen-carboxymethyl chitosan composite hydrogel to the zirconium cross-linking solution was 1:5; finally, the zirconium was The fixed composite hydrogel was placed in 20% titanium sulfate solution and mineralized at 40°C for 72 hours to obtain high-strength titanium mineralized collagen hydrogel, zirconium cross-linked collagen-carboxymethyl chitosan composite hydrogel. The volume ratio of glue to titanyl sulfate solution is 1:10.

The storage modulus of the titanium mineralized collagen hydrogel prepared in this example and suitable for bone repair materials is 264.5 kPa.

Example 3:

Dissolve collagen sponge and carboxymethyl chitosan in 200mmol/L disodium hydrogen phosphate solution respectively to prepare collagen solution and carboxymethyl chitosan solution; the mass concentration of collagen solution is 0.4%, and the mass concentration of carboxymethyl chitosan is 0.4%. The mass concentration of the sugar solution is 0.1%. The volumes of the collagen solution and the carboxymethyl chitosan solution are the same. The pH value of each solution during the dissolution process is controlled to be 7 and the temperature is 2°C. Slowly add the carboxymethyl chitosan solution dropwise to the stirring collagen solution at 2°C, and stir for 12 hours to prepare a collagen-carboxymethyl chitosan composite solution. Subsequently, the composite solution was placed at 30°C for self-assembly for 3 hours to prepare a collagen-carboxymethyl chitosan composite hydrogel. Dissolve trisodium citrate and zirconium sulfate tetrahydrate in deionized water, and prepare a zirconium cross-linking solution with a mass concentration of zirconium sulfate tetrahydrate of 0.1%, in which the mass ratio of trisodium citrate to zirconium sulfate tetrahydrate is: 1:1. The composite hydrogel was placed in the zirconium cross-linking solution and fixed with zirconium at 25°C for 48 hours. The volume ratio of the collagen-carboxymethyl chitosan composite hydrogel to the zirconium cross-linking solution was 1:1; finally, the zirconium was The fixed composite hydrogel was placed in 0.1% titanyl sulfate solution and mineralized at 25°C for 12 hours to obtain high-strength titanium mineralized collagen hydrogel, zirconium cross-linked collagen-carboxymethyl chitosan composite hydrogel. The volume ratio of glue to titanyl sulfate solution is 1:2.

The storage modulus of the titanium mineralized collagen hydrogel prepared in this example and suitable for bone repair materials is 17.8 kPa.

Example 4:

Dissolve collagen sponge and carboxymethyl chitosan in 100mmol/L sodium chloride solution respectively to prepare collagen solution and carboxymethyl chitosan solution; the mass concentration of collagen solution is 1.2%, and carboxymethyl chitosan The mass concentration of the solution is 2.4%. The volumes of the collagen solution and the carboxymethyl chitosan solution are the same. The pH value of each solution during the dissolution process is controlled to be 7.4 and the temperature is 7°C. Slowly add the carboxymethyl chitosan solution dropwise to the stirring collagen solution at 7°C, and stir for 18 hours to prepare a collagen-carboxymethyl chitosan composite solution. Subsequently, the composite solution was placed at 32°C for self-assembly for 3 h to prepare a collagen-carboxymethyl chitosan composite hydrogel. Dissolve trisodium citrate and zirconium sulfate tetrahydrate in deionized water, and prepare a zirconium cross-linking solution with a mass concentration of zirconium sulfate tetrahydrate of 1%, in which the mass ratio of trisodium citrate to zirconium sulfate tetrahydrate is 1:2.5. The composite hydrogel was placed in the zirconium cross-linking solution and fixed with zirconium at 30°C for 84 hours. The volume ratio of the collagen-carboxymethyl chitosan composite hydrogel to the zirconium cross-linking solution was 1:4; finally, the zirconium was The fixed composite hydrogel was placed in 10% titanyl sulfate solution and mineralized at 30°C for 48 hours to obtain high-strength titanium mineralized collagen hydrogel, zirconium cross-linked collagen-carboxymethyl chitosan composite hydrogel. The volume ratio of glue to titanium sulfate solution is 1:4.

The storage modulus of the titanium mineralized collagen hydrogel suitable for bone repair materials prepared in this embodiment is 137.9 kPa.

Example 5:

Dissolve collagen sponge and carboxymethyl chitosan in 160mmol/L sodium dihydrogen phosphate solution respectively to prepare collagen solution and carboxymethyl chitosan solution; the mass concentration of collagen solution is 1.5%, and the mass concentration of carboxymethyl chitosan is 1.5%. The mass concentration of the sugar solution is 6%. The volumes of the collagen solution and the carboxymethyl chitosan solution are the same. The pH value of each solution during the dissolution process is controlled to be 7.6 and the temperature is 8°C. Slowly add the carboxymethyl chitosan solution dropwise to the stirring collagen solution at 8°C, and stir for 20 hours to prepare a collagen-carboxymethyl chitosan composite solution. Subsequently, the composite solution was placed at 35°C for self-assembly for 3 h to prepare a collagen-carboxymethyl chitosan composite hydrogel. Dissolve trisodium citrate and zirconium sulfate tetrahydrate in deionized water, and prepare a zirconium cross-linking solution with a mass concentration of zirconium sulfate tetrahydrate of 3%, in which the mass ratio of trisodium citrate to zirconium sulfate tetrahydrate is 1:3. The composite hydrogel was placed in the zirconium cross-linking solution and fixed with zirconium at 35°C for 60 hours. The volume ratio of the collagen-carboxymethyl chitosan composite hydrogel to the zirconium cross-linking solution was 1:7; finally, the zirconium was The fixed composite hydrogel was placed in 15% titanyl sulfate solution and mineralized at 35°C for 60 hours to obtain high-strength titanium mineralized collagen hydrogel, zirconium cross-linked collagen-carboxymethyl chitosan composite hydrogel. The volume ratio of glue to titanyl sulfate solution is 1:3.

The storage modulus of the titanium mineralized collagen hydrogel prepared in this example and suitable for bone repair materials is 151.5 kPa.

Example 6

Dissolve collagen sponge and carboxymethyl chitosan in 125mmol/L sodium dihydrogen phosphate solution respectively to prepare collagen solution and carboxymethyl chitosan solution; the mass concentration of collagen solution is 1.8%, and carboxymethyl chitosan solution is 1.8%. The mass concentration of the sugar solution is 5.2%. The volumes of the collagen solution and the carboxymethyl chitosan solution are the same. The pH value of each solution during the dissolution process is controlled to be 7 and the temperature is 5°C. Slowly add the carboxymethyl chitosan solution dropwise to the stirring collagen solution at 5°C, and stir for 20 hours to prepare a collagen-carboxymethyl chitosan composite solution. Subsequently, the composite solution was placed at 35°C for self-assembly for 3 h to prepare a collagen-carboxymethyl chitosan composite hydrogel. Dissolve trisodium citrate and zirconium sulfate tetrahydrate in deionized water, and prepare a zirconium cross-linking solution with a mass concentration of zirconium sulfate tetrahydrate of 3%, in which the mass ratio of trisodium citrate to zirconium sulfate tetrahydrate is 1:3.5. The composite hydrogel was placed in the zirconium cross-linking solution and fixed with zirconium at 35°C for 65 hours. The volume ratio of the collagen-carboxymethyl chitosan composite hydrogel to the zirconium cross-linking solution was 1:7; finally, the zirconium was The fixed composite hydrogel was placed in 8% titanyl sulfate solution and mineralized at 30°C for 70 hours to obtain high-strength titanium mineralized collagen hydrogel, zirconium cross-linked collagen-carboxymethyl chitosan composite hydrogel. The volume ratio of glue to titanyl sulfate solution is 1:7.5.

The storage modulus of the titanium mineralized collagen hydrogel suitable for bone repair materials prepared in this embodiment is 233.7 kPa.

Example 7

Dissolve collagen sponge and carboxymethyl chitosan in 120mmol/L disodium hydrogen phosphate solution respectively to prepare collagen solution and carboxymethyl chitosan solution; the mass concentration of collagen solution is 1%, and carboxymethyl chitosan solution is 1%. The mass concentration of the sugar solution is 1%, the volumes of the collagen solution and the carboxymethyl chitosan solution are the same, the pH value of each solution during the dissolution process is controlled to 7.1, and the temperature is 2°C. Slowly add the carboxymethyl chitosan solution dropwise to the stirring collagen solution at 2°C, and stir for 16 hours to prepare a collagen-carboxymethyl chitosan composite solution. Subsequently, the composite solution was placed at 36°C for self-assembly for 3 h to prepare a collagen-carboxymethyl chitosan composite hydrogel. Dissolve trisodium citrate and zirconium sulfate tetrahydrate in deionized water, and prepare a zirconium cross-linking solution with a mass concentration of zirconium sulfate tetrahydrate of 0.5%, in which the mass ratio of trisodium citrate to zirconium sulfate tetrahydrate is 1:2. The composite hydrogel was placed in the zirconium cross-linking solution and fixed with zirconium at 30°C for 60 hours. The volume ratio of the collagen-carboxymethyl chitosan composite hydrogel to the zirconium cross-linking solution was 1:5; finally, the zirconium was The fixed composite hydrogel was placed in 0.5% titanyl sulfate solution and mineralized at 30°C for 48 hours to obtain high-strength titanium mineralized collagen hydrogel, zirconium cross-linked collagen-carboxymethyl chitosan composite hydrogel. The volume ratio of glue to titanyl sulfate solution is 1:5.

The storage modulus of the titanium mineralized collagen hydrogel prepared in this example and suitable for bone repair materials is 73.4 kPa.

Example 8

Dissolve collagen sponge and carboxymethyl chitosan in a sodium salt solution of 20mmol/L disodium hydrogen phosphate/sodium dihydrogen phosphate and 100mmol/L sodium chloride, respectively, to prepare a collagen solution and a carboxymethyl chitosan solution. ; The mass concentration of the collagen solution is 0.6%, and the mass concentration of the carboxymethyl chitosan solution is 0.3%. The volumes of the collagen solution and the carboxymethyl chitosan solution are the same. The pH value of each solution during the dissolution process is controlled to 7.5. The temperature is 5℃. Slowly add the carboxymethyl chitosan solution dropwise to the stirring collagen solution at 5°C, and stir for 24 hours to prepare a collagen-carboxymethyl chitosan composite solution. Subsequently, the composite solution was placed at 37°C for self-assembly for 3 hours to prepare a collagen-carboxymethyl chitosan composite hydrogel. Dissolve trisodium citrate and zirconium sulfate tetrahydrate in deionized water, and prepare a zirconium cross-linking solution with a mass concentration of zirconium sulfate tetrahydrate of 1.2%, in which the mass ratio of trisodium citrate to zirconium sulfate tetrahydrate is 1:2.5. The composite hydrogel was placed in the zirconium cross-linking solution and fixed with zirconium at 35°C for 70 hours. The volume ratio of the collagen-carboxymethyl chitosan composite hydrogel to the zirconium cross-linking solution was 1:7.5; finally, the zirconium was The fixed composite hydrogel was placed in 2.5% titanyl sulfate solution and mineralized at 35°C for 55 hours to obtain high-strength titanium mineralized collagen hydrogel, zirconium cross-linked collagen-carboxymethyl chitosan composite hydrogel. The volume ratio of glue to titanyl sulfate solution is 1:8.

The storage modulus of the titanium mineralized collagen hydrogel prepared in this example and suitable for bone repair materials is 56.2 kPa.

Example 9

Dissolve the collagen sponge and carboxymethyl chitosan in a sodium salt solution of 160 mmol/L sodium chloride to prepare a collagen solution and a carboxymethyl chitosan solution; the mass concentration of the collagen solution is 0.8%, and the carboxymethyl chitosan solution is 0.8%. The mass concentration of the chitosan solution is 1.5%. The volumes of the collagen solution and the carboxymethyl chitosan solution are the same. The pH value of each solution during the dissolution process is controlled to be 8 and the temperature is 8°C. Slowly add the carboxymethyl chitosan solution dropwise to the stirring collagen solution at 8°C, and stir for 24 hours to prepare a collagen-carboxymethyl chitosan composite solution. Subsequently, the composite solution was placed at 34°C for self-assembly for 3 h to prepare a collagen-carboxymethyl chitosan composite hydrogel. Dissolve trisodium citrate and zirconium sulfate tetrahydrate in deionized water, and prepare a zirconium cross-linking solution with a mass concentration of zirconium sulfate tetrahydrate of 2%, in which the mass ratio of trisodium citrate to zirconium sulfate tetrahydrate is 1:4.5. The composite hydrogel was placed in the zirconium cross-linking solution and fixed with zirconium at 40°C for 80 hours. The volume ratio of the collagen-carboxymethyl chitosan composite hydrogel to the zirconium cross-linking solution was 1:10; finally, the zirconium was fixed The fixed composite hydrogel was placed in 5% titanyl sulfate solution and mineralized at 37°C for 60 hours to obtain high-strength titanium mineralized collagen hydrogel, zirconium cross-linked collagen-carboxymethyl chitosan composite hydrogel. The volume ratio of glue to titanium sulfate solution is 1:3.5.

The storage modulus of the titanium mineralized collagen hydrogel prepared in this example and suitable for bone repair materials is 102.6 kPa.

Example 10

Dissolve collagen sponge and carboxymethyl chitosan in 100mmol/L disodium hydrogen phosphate/sodium dihydrogen phosphate solution to prepare collagen solution and carboxymethyl chitosan solution; the mass concentration of the collagen solution is 1%. The mass concentration of the carboxymethyl chitosan solution is 2%, the volumes of the collagen solution and the carboxymethyl chitosan solution are the same, and the pH value of each solution during the dissolution process is controlled to be 8 and the temperature is 10°C. Slowly add the carboxymethyl chitosan solution dropwise to the stirring collagen solution at 10°C, and stir for 19 hours to prepare a collagen-carboxymethyl chitosan composite solution. Subsequently, the composite solution was placed at 33°C for self-assembly for 3 h to obtain a collagen-carboxymethyl chitosan composite hydrogel. Dissolve trisodium citrate and zirconium sulfate tetrahydrate in deionized water, and prepare a zirconium cross-linking solution with a mass concentration of zirconium sulfate tetrahydrate of 2.5%, in which the mass ratio of trisodium citrate to zirconium sulfate tetrahydrate is 1:5. The composite hydrogel was placed in the zirconium cross-linking solution and fixed with zirconium at 36°C for 96 hours. The volume ratio of the collagen-carboxymethyl chitosan composite hydrogel to the zirconium cross-linking solution was 1:4; finally, the zirconium was The fixed composite hydrogel was placed in 10% titanyl sulfate solution and mineralized at 40°C for 72 hours to obtain high-strength titanium mineralized collagen hydrogel, zirconium cross-linked collagen-carboxymethyl chitosan composite hydrogel. The volume ratio of glue to titanyl sulfate solution is 1:6.5.

The storage modulus of the titanium mineralized collagen hydrogel prepared in this example and suitable for bone repair materials is 91.4 kPa.

Figure 2 is a scanning electron microscope image of pure collagen (product of Comparative Example 1) and titanium mineralized collagen hydrogel (product of Example 1). It can be observed from the figure that the fiber surface in pure collagen hydrogel is smooth, while the fiber surface in mineralized collagen hydrogel is wrapped with a large number of spherical TiO ₂ .

Figure 3 shows the storage modulus of pure collagen (product of Comparative Example 1) and titanium mineralized collagen hydrogel (product of Example 1). The storage modulus of titanium mineralized collagen hydrogel (120.0kPa) is much larger than that of unmineralized pure collagen hydrogel (90.8Pa), about 1320 times, indicating that the introduction of TiO ₂ gives it excellent mechanical properties.

Among them, the specific testing process of storage modulus in this application is:

Place the hydrogel in the flat plate fixture of the rheometer (diameter 20mm, plate spacing 1.0mm), perform a stress-strain scan on it, determine the linear viscoelastic region of the hydrogel and select the appropriate strain size within the region :1%. Then a dynamic frequency sweep was performed in strain control mode to obtain the storage modulus. The test temperature is 25±0.1℃, and the frequency range is 0.01~10Hz.

Storage modulus, also known as elastic modulus, is an index that measures the ease of elastic deformation of a material. The greater the value, the greater the stress that causes the material to undergo a certain elastic deformation, that is, the greater the stiffness of the material, that is, under a certain Under the action of stress, the smaller the elastic deformation occurs. Generally expressed by G', the unit of storage modulus is Pascal (Pa), kilopascal (kPa) or megapascal (MPa). The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be made. regarded as the protection scope of the present invention.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and do not limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that The technical solution of the present invention may be modified or equivalently substituted without departing from the essence and scope of the technical solution of the present invention.

Claims (10)

1. A method for preparing a titanium-structured collagen hydrogel suitable for a bone repair material, which is characterized by comprising the following steps:

s1: respectively dissolving collagen sponge and carboxymethyl chitosan in a sodium salt solution to prepare a collagen solution and a carboxymethyl chitosan solution;

s2: dropwise adding the carboxymethyl chitosan solution into the collagen solution, and stirring for reaction to obtain a collagen-carboxymethyl chitosan composite solution;

s3: self-assembling the collagen-carboxymethyl chitosan composite solution into fibers to prepare the collagen-carboxymethyl chitosan composite hydrogel;

s4: placing the collagen-carboxymethyl chitosan composite hydrogel into zirconium crosslinking liquid for reaction to prepare zirconium crosslinked collagen-carboxymethyl chitosan composite hydrogel;

s5: and placing the zirconium crosslinked collagen-carboxymethyl chitosan composite hydrogel in a titanyl sulfate solution for mineralization reaction to prepare the titanium-mineral collagen hydrogel suitable for the bone repair material.

2. The method for preparing a titanium-structured collagen hydrogel for use in a bone repair material according to claim 1, wherein in step S1, the salt concentration in the sodium salt solution is 100 to 200mmol/L; the mass concentration of the collagen solution is 0.4% -2%.

3. The method for preparing the titanium-structured collagen hydrogel for bone repair materials according to claim 1, wherein in the step S2, the mass ratio of collagen to carboxymethyl chitosan in the collagen-carboxymethyl chitosan composite solution is 1 (0.25-4); controlling the pH value of the reaction system in the step S2 to be 7-8, the reaction temperature to be 2-10 ℃ and the stirring time to be 12-24 h.

4. The method for preparing the titanium-base collagen hydrogel for the bone repair material according to claim 1, wherein in the step S3, the collagen-carboxymethyl chitosan composite solution is subjected to self-assembly at 30-37 ℃ to prepare the collagen-carboxymethyl chitosan composite hydrogel.

5. The method for preparing a titanium-structured collagen hydrogel for use in a bone repair material according to claim 1, wherein in step S4, the zirconium cross-linking liquid is prepared by dissolving trisodium citrate and zirconium sulfate tetrahydrate in water, wherein the mass ratio of trisodium citrate to zirconium sulfate tetrahydrate is 1 (1-5); the mass concentration of the zirconium sulfate tetrahydrate in the zirconium crosslinking liquid is 0.1-5%.

6. The method for preparing the titanium-structured collagen hydrogel for bone repair materials according to claim 1, wherein in the step S4, the volume ratio of the collagen-carboxymethyl chitosan composite hydrogel to the zirconium cross-linking solution is 1 (1-10); the reaction temperature in the step S4 is 25-40 ℃ and the reaction time is 48-96 h.

7. The method for preparing a titanium-oxide-sulfate-type collagen hydrogel for use as a bone repair material according to claim 1, wherein in step S5, the mass concentration of the titanium-oxide-sulfate solution is 0.1% to 20%; the volume ratio of the zirconium crosslinked collagen-carboxymethyl chitosan composite hydrogel to the titanyl sulfate solution is 1 (1-10).

8. The method for preparing a titanium-structured collagen hydrogel for use in a bone repair material according to claim 1, wherein in step S5, the mineralization process specifically comprises: mixing zirconium crosslinked collagen-carboxymethyl chitosan composite hydrogel with a titanyl sulfate solution, and then mineralizing the mixed solution at 25-40 ℃ for 24-72 h to prepare the titanium-mineral collagen hydrogel suitable for bone repair materials.

9. A titanium-structured collagen hydrogel suitable for use in a bone repair material, characterized in that it is produced by the method according to any one of claims 1 to 8, and has a storage modulus of 17.8 to 264.5kPa.

10. A bone repair material comprising a titanium-bearing collagen hydrogel suitable for use in a bone repair material as claimed in claim 9.