A titanium mineralized collagen hydrogel suitable for bone repair material and its preparation method

Abstract

The present invention discloses a titanium mineralized collagen hydrogel suitable for bone repair materials and a preparation method thereof. The present invention uses natural collagen, the main structural protein constituting cartilage, as a template, natural macromolecular carboxymethyl chitosan as an inducer, and titanium oxysulfate solution as a precursor solution. Based on the principle of biomimetic mineralization, the present invention drives titanium oxysulfate to hydrolyze and condense into titanium dioxide (TiO2) and deposit on collagen fibers. The two produce a strong interfacial interaction to obtain a high-strength titanium mineralized collagen hydrogel. The prepared mineralized hydrogel improves the mechanical properties of pure collagen hydrogel, while giving it advantages such as good bone conduction ability, biocompatibility, hemostasis and antibacterial properties. The hydrogel can be used as a bone repair material alone or in combination with other biological factors and drugs.

Description

Technical Field

[0001] 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 Art

[0002] Bone tissue damage is a serious health problem worldwide. In current clinical practice, bone transplantation is the most common method for bone repair, primarily including autologous bone, allogeneic bone, and tissue-engineered artificial bone transplantation. However, autologous bone transplantation has drawbacks such as limited bone supply and donor-site complications. Allogeneic bone transplantation has limited osteoinductivity, immunogenicity, and immune response. These limitations have made the development of bone repair materials in bone tissue engineering a current research hotspot, providing a new therapeutic strategy for bone damage. Ideal scaffold materials should possess good biocompatibility, be non-toxic, low-cost, non-carcinogenic, and possess excellent osteoconductive properties. Hydrogels possess a three-dimensional reticular structure similar to the extracellular matrix. Their mild environment protects cells and promotes their uniform distribution in three dimensions, providing structural support for the defect site and enabling repair of bone defects through intrinsic healing mechanisms. More importantly, by utilizing the solution-to-gel transition property, they can be injected and implanted for repairing complex geometric tissues, reducing surgical trauma through in situ molding.

[0003] Collagen is the main structural protein that makes up cartilage and bone. It is an essential nutrient for bone tissue metabolism, 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, materials constructed from collagen fibers have a microstructure very similar to that of collagen in tissues. They have the advantages of slow degradation, high integration with surrounding tissues, and inhibition of inflammation, and can more effectively exert their biological functions. Collagen hydrogels formed by collagen fiber-coated aqueous solutions are natural hydrogels with high water content, rapid swelling, stable structure, and good biocompatibility. They can be used as biomimetic scaffolds in both cartilage and bone tissue engineering and have broad research and development prospects. However, the mechanical strength of pure collagen hydrogels is not high and cannot be directly applied in tissue engineering. Therefore, modifying collagen hydrogels to improve their mechanical strength is a current research hotspot.

[0004] At present, there are three main methods to improve the strength of collagen hydrogels: 1) Physical method: no exogenous substances are introduced, which prevents exogenous toxic and harmful chemicals from entering the collagen hydrogel, but it is difficult to obtain a uniform and consistent cross-linked 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 blended materials are retained, the improvement in its mechanical strength is not obvious.

[0005] Biomineralization is a process in which biomacromolecules and certain inducing factors regulate the orderly nucleation and growth of inorganic minerals under certain physiological conditions, producing biominerals with fine structures. The biomineralized materials formed by this process are organic-inorganic nanohybrid materials found in nature, with a multi-level structural organization ranging from nanometers and micrometers to macroscales, and possess high hardness and fracture resistance. Examples include hard tissues such as human bones and teeth. Bone tissue is a complex biomineralized material, primarily formed through biomineralization of collagen, hydroxyapatite, and non-collagenous proteins. Collagen acts as a scaffold, imparting elasticity and toughness to the bone, while hydroxyapatite and collagen fibrils exhibit organic-inorganic interface recognition and intercalation, providing stiffness. Furthermore, the multi-fibrillar structure of collagen fibrils lays the foundation for the multi-level micro- and nanostructure of bone tissue. This strict hierarchical structure provides bone tissue with superior mechanical properties. Inspired by this, the use of biomimetic mineralization technology to combine hydroxyapatite or other inorganic substances with collagen to prepare bone repair materials has become a research hotspot. However, the prepared materials are mostly membrane-like or sponge-like, and few use this technology to prepare high-strength collagen hydrogels while retaining the fiber structure of collagen in the hydrogel. Summary of the Invention

[0006] 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.

[0007] The present invention is achieved through the following technical solutions:

[0008] A method for preparing titanium mineralized collagen hydrogel suitable for bone repair material comprises the following steps:

[0009] S1: Dissolving collagen sponge and carboxymethyl chitosan in sodium salt solution to prepare collagen solution and carboxymethyl chitosan solution respectively;

[0010] S2: adding the carboxymethyl chitosan solution dropwise to the collagen solution, stirring to react, and obtaining a collagen-carboxymethyl chitosan composite solution;

[0011] S3: allowing the collagen-carboxymethyl chitosan composite solution to self-assemble into fibers to prepare a collagen-carboxymethyl chitosan composite hydrogel;

[0012] S4: placing the collagen-carboxymethyl chitosan composite hydrogel in a zirconium cross-linking solution to react, thereby obtaining a zirconium-cross-linked collagen-carboxymethyl chitosan composite hydrogel;

[0013] S5: placing the zirconium-crosslinked collagen-carboxymethyl chitosan composite hydrogel in a titanyl sulfate solution to carry out a mineralization reaction, thereby obtaining the titanium-mineralized collagen hydrogel suitable for bone repair material.

[0014] Preferably, in step S1, the salt concentration of the sodium salt solution is 100-200 mmol / L; and the mass concentration of the collagen solution is 0.4%-2%.

[0015] 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 to be 7-8, and the reaction temperature is controlled to be 2-10°C.

[0016] Preferably, in step S3, the collagen-carboxymethyl chitosan composite solution is placed at 30-37° C. for self-assembly to obtain the collagen-carboxymethyl chitosan composite hydrogel.

[0017] Preferably, 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); and the mass concentration of zirconium sulfate tetrahydrate in the zirconium cross-linking liquid is 0.1%-5%.

[0018] Preferably, in step S4, the volume ratio of the collagen-carboxymethyl chitosan composite hydrogel to the zirconium cross-linking liquid is 1:(1-10); the reaction temperature in step S4 is 25-40° C., and the reaction time is 48-96 h.

[0019] Preferably, in step S5, the mass concentration of the titanyl sulfate solution is 0.1% to 20%; and the volume ratio of the zirconium-crosslinked collagen-carboxymethyl chitosan composite hydrogel to the titanyl sulfate solution is 1:(1 to 10).

[0020] Preferably, in step S5, the mineralization process is specifically as follows: after mixing the zirconium-crosslinked collagen-carboxymethyl chitosan composite hydrogel with the titanium oxysulfate solution, the mixture is placed at 25 to 40° C. for mineralization for 24 to 72 hours to obtain the titanium mineralized collagen hydrogel suitable for bone repair material.

[0021] 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 for bone repair is 17.8-264.5 kPa.

[0022] A bone repair material comprises the above-mentioned titanium mineralized collagen hydrogel suitable for bone repair material.

[0023] Compared with the prior art, the present invention has the following beneficial technical effects:

[0024] A titanium-mineralized collagen hydrogel suitable for bone repair materials and a preparation method thereof, using collagen fibers that self-assemble into a multi-level structure as a template, natural macromolecular carboxymethyl chitosan as an inducer, and a titanium oxysulfate solution as a precursor solution. Based on the principle of biomimetic mineralization, titanium oxysulfate is driven to hydrolyze and condense inside and on the surface of the fibers to undergo mineralization, forming a composite hydrogel of TiO2 and collagen fibers. This process creates a strong interfacial interaction between the inorganic matter and the collagen fibers, thereby giving the collagen hydrogel better mechanical properties; TiO2 has good biocompatibility, bone conduction ability, and excellent mechanical properties, can induce hydroxyapatite deposition, and is an active ingredient for bone integration in the body, giving the hydrogel good bone conduction ability and bone repair function. In addition, carboxymethyl chitosan is an amphoteric polyelectrolyte that not only induces the hydrolysis and condensation of titanium oxysulfate, but also gives the composite hydrogel good hemostatic and antibacterial properties, and enhances the material's role in promoting the regeneration and repair of articular cartilage. At the same time, during the preparation process, the collagen-carboxymethyl chitosan composite hydrogel was protected by a zirconium cross-linking liquid and then placed in a titanyl sulfate solution for titanium mineralization, which effectively prevented the collagen fibers from being destroyed under strong acid conditions and effectively maintained the multi-level fiber structure of the hydrogel material.

[0025] Furthermore, the salt concentration in the sodium salt solution is 100-200 mmol / L, and the sodium ions and other anions perform charge shielding on the collagen and carboxymethyl chitosan, thereby preventing coagulation after the collagen and carboxymethyl chitosan are mixed; the mass concentration of the collagen solution is 0.4%-2%. If the concentration is too low, the fibers formed by self-assembly are immature, while if the concentration is too high, the viscosity of the system is too high, and 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 viscosity of the system too high, effectively meeting the experimental requirements.

[0026] 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 blended solution will be poor, which is not conducive to obtaining a product with good performance. The pH value of the reaction system in step S2 is controlled to be 7-8. This pH value can effectively realize the subsequent self-assembly process, and at this pH value, the electrostatic charge carried by collagen is the least. Above or below this range, the charge on the surface of collagen is too high, and the electrostatic force between collagen and carboxymethyl chitosan is too strong, and the two are prone to coagulation. The reaction temperature of the reaction system in step S2 is controlled to be 2-10°C. This temperature can effectively maintain the triple helical structure of collagen, i.e., biological activity, and prevent collagen from self-assembling when blended with carboxymethyl chitosan.

[0027] Furthermore, in step S3, the collagen-carboxymethyl chitosan composite solution is placed at 30-37° C. for self-assembly to obtain the collagen-carboxymethyl chitosan composite hydrogel. This temperature effectively realizes the self-assembly process of collagen.

[0028] Furthermore, in step S4, the zirconium cross-linking liquid is prepared by dissolving trisodium citrate and zirconium sulfate tetrahydrate in water. Zirconium cross-linking effectively improves the acid resistance of the collagen-carboxymethyl chitosan composite hydrogel. 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 fiber structure assembled by the collagen molecules will be destroyed, the advantages of the collagen fibers will be lost, and the hydrogel may even be dissociated into a solution and the modified hydrogel material cannot be prepared. Trisodium citrate mainly coordinates with zirconium sulfate. After being added, the pH value of the zirconium sulfate solution can be increased, thereby preventing the strong acidity of the pure zirconium sulfate solution from damaging collagen fibers. The mass ratio of trisodium citrate to zirconium sulfate tetrahydrate 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 plays an effective protective role on the fiber structure in the collagen-carboxymethyl chitosan composite hydrogel. When the concentration of zirconium sulfate tetrahydrate is too low, the purpose of protection and fixation cannot be achieved. When the concentration of the zirconium sulfate solution is too high, the system becomes too acidic, requiring the addition of a large amount of trisodium citrate, resulting in a waste of raw materials and an increase in impurities in the system.

[0029] Furthermore, in step S4, the volume ratio of the collagen-carboxymethyl chitosan composite hydrogel to the zirconium crosslinking solution is 1:(1-10), so that the zirconium crosslinking effectively protects the collagen-carboxymethyl chitosan composite hydrogel, preventing it from dissolving during the later mineralization process. In addition, the reaction temperature in step S4 is 25-40°C, and the reaction time is 48-96 hours. These reaction parameters effectively improve the crosslinking efficiency of zirconium and the collagen-carboxymethyl chitosan composite hydrogel, thereby improving the acid resistance of the hydrogel.

[0030] Furthermore, in step S5, the mass concentration of the titanyl 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 to 10), thereby achieving full functionalization of the interior and surface of the collagen-carboxymethyl chitosan composite hydrogel by TiO2. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0032] Figure 1 Schematic diagram of a process for preparing a titanium mineralized collagen hydrogel suitable for bone repair materials in the present invention;

[0033] Figure 2 The following are scanning electron microscope (SEM) images 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.

[0034] Figure 3 This is a comparison chart of the storage modulus of 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 titanium mineralized collagen hydrogel suitable for bone repair materials in the present invention, where a-collagen hydrogel, b-high-strength titanium mineralized collagen hydrogel. DETAILED DESCRIPTION

[0035] To facilitate understanding of the features and effects of the present invention by those skilled in the art, the following provides a general description and definition of the terms and expressions used in the specification and claims. Unless otherwise indicated, all technical and scientific terms used herein have the ordinary 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.

[0036] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0037] Herein, all features such as values, amounts, amounts, and concentrations defined in numerical ranges or percentage ranges are for brevity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to include and specifically disclose all possible subranges and individual values ​​within the range (including integers and fractions).

[0038] In this document, unless otherwise specified, “include,” “including,” “contains,” “has” or similar terms cover the meanings of “consisting of” and “mainly consisting of,” for example, “A includes a” covers the meanings of “A includes a and other” and “A only includes a.”

[0039] In this document, for the sake of brevity, not all possible combinations of the various technical features in each embodiment or example are described. Therefore, as long as there are no contradictions in the combination of these technical features, the various technical features 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.

[0040] like Figure 1 As shown, the present invention provides a titanium mineralized collagen hydrogel suitable for bone repair materials and a preparation method thereof, specifically:

[0041] S1: Dissolving collagen sponge and carboxymethyl chitosan in a sodium salt solution 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%, respectively. During the dissolution process, the pH value of each solution is controlled to be 7 to 8 and the temperature to be 2 to 10°C.

[0042] The salt concentration in the sodium salt solution is 100 to 200 mmol / L, more preferably 120 to 160 mmol / L. The sodium salt is used to shield the charge of collagen and carboxymethyl chitosan and prevent precipitation after the collagen and carboxymethyl chitosan are mixed. 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 are immature, while if the concentration is too high, the viscosity of the system is 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 viscosity of the system too high, effectively meeting the experimental requirements. The mass concentration of the carboxymethyl chitosan solution is 0.1% to 8%, more preferably 0.3% to 3.2%.

[0043] In addition, the sodium salt solution is selected from at least one of disodium hydrogen phosphate, sodium dihydrogen phosphate, or sodium chloride. The reasons for choosing sodium salt here are that first, collagen is insoluble in deionized water, second, sodium salt can shield the charges of collagen and carboxymethyl chitosan, preventing them from precipitation, and in addition, the participation of salt is required for collagen self-assembly.

[0044] S2: slowly adding the carboxymethyl chitosan solution dropwise to the collagen solution, stirring to react, and obtaining a collagen-carboxymethyl chitosan composite solution, wherein the collagen and the carboxymethyl chitosan are bonded via electrostatic and hydrogen bonding.

[0045] Among them, the dry weight 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). A ratio that is too high or too low will lead to poor compatibility of the blended solution, which is not conducive to obtaining a product with good performance; in addition, the amount of carboxymethyl chitosan is related to the mineralization effect, because the subsequent mineralization process of titanyl sulfate mainly depends on the induction effect of carboxymethyl chitosan. It was found in the experiment that when carboxymethyl chitosan was not added, the hydrolysis and condensation process of titanyl sulfate was very slow, resulting in a long mineralization time and too little titanium dioxide deposition. The pH of the reaction system in this step is controlled to 7-8, more preferably 7.2-7.6. This pH value can effectively achieve the self-assembly process. At this pH value, the charge on the collagen surface is minimal. Above or below this range, the charge on the collagen surface is too high, and the electrostatic interaction between the collagen and the carboxymethyl chitosan is too strong, which easily causes precipitation. The reaction temperature is controlled to 2-10°C to effectively maintain the stability of the collagen and prevent self-assembly when blending with the carboxymethyl chitosan. The stirring reaction is controlled to 12-24 hours, more preferably 16-20 hours, to ensure that the collagen and carboxymethyl chitosan are more fully combined.

[0046] S3: The collagen-carboxymethyl chitosan composite solution is placed at 30-37° C. for self-assembly for 3 hours to obtain 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.

[0047] S4: The collagen-carboxymethyl chitosan composite hydrogel is mixed with a zirconium cross-linking solution, stirred for reaction, and a zirconium-cross-linked collagen-carboxymethyl chitosan composite hydrogel is obtained. The reason why the collagen-carboxymethyl chitosan composite hydrogel needs to be zirconium-cross-linked here is mainly because the titanyl sulfate solution is highly acidic. If the collagen-carboxymethyl chitosan composite hydrogel is directly placed in the titanyl sulfate solution, the collagen fiber structure will be destroyed, losing the advantages of the collagen fibers, and even the hydrogel will be dissociated into a solution, and the modified hydrogel material cannot be obtained. Therefore, the zirconium cross-linking process here plays an effective protective role for the collagen-carboxymethyl chitosan composite hydrogel.

[0048] The zirconium cross-linking liquid is prepared by dissolving trisodium citrate and zirconium sulfate tetrahydrate in deionized water at room temperature, wherein the mass ratio of trisodium citrate to zirconium sulfate tetrahydrate is 1:(1-5), more preferably 1:(2-3.5). Here, trisodium citrate mainly coordinates zirconium sulfate, and after addition, the pH value of the zirconium sulfate solution can be increased, thereby avoiding damage to the fiber. The mass concentration of zirconium sulfate tetrahydrate in the zirconium cross-linking liquid is 0.1% to 5%, more preferably 0.5% to 2.5%. The zirconium cross-linking process mainly plays an effective protective role on the collagen-carboxymethyl chitosan composite hydrogel; when the concentration of zirconium sulfate tetrahydrate is too low, the purpose of protection and fixation cannot be achieved, and when the concentration of the zirconium sulfate solution is too high, the system is too acidic, and a large amount of trisodium citrate needs to be added, resulting in a waste of raw materials and an increase in impurities in the system.

[0049] The volume ratio of the collagen-carboxymethyl chitosan composite hydrogel to the zirconium cross-linking liquid is 1:(1-10); the reaction temperature in step S4 is 25-40°C, more preferably 30-37°C, and the reaction time is 48-96h, more preferably 60-80h. These reaction parameters effectively improve the cross-linking effect between zirconium and the collagen-carboxymethyl chitosan composite hydrogel, thereby improving the acid resistance of the hydrogel.

[0050] S5: mixing the zirconium-crosslinked collagen-carboxymethyl chitosan composite hydrogel with a titanyl sulfate solution, stirring the mixture for reaction and performing mineralization to obtain the titanium-mineralized collagen-carboxymethyl chitosan composite hydrogel; the mineralization process is a process in which titanyl sulfate is converted into TiO2.

[0051] Among them, the preparation process of the titanyl sulfate solution is specifically as follows: dissolving titanyl sulfate in deionized water at room temperature to obtain a titanyl sulfate solution with a mass concentration of 0.1% to 20%, and more preferably the mass concentration of the titanyl sulfate solution is 1% to 10%; the volume ratio of the zirconium-crosslinked collagen-carboxymethyl chitosan composite hydrogel to the titanyl sulfate solution is 1:(1 to 10).

[0052] The specific mineralization process is as follows: after mixing the zirconium-crosslinked collagen-carboxymethyl chitosan composite hydrogel with the titanium sulfate solution, the mixture is placed at 25-40°C for mineralization for 24-72 hours to obtain the titanium mineralized collagen-carboxymethyl chitosan composite hydrogel. Here, the mineralization temperature is more preferably 30-37°C, and the mineralization time is more preferably 48-60 hours.

[0053] The collagen sponge in this application is prepared by the following method: fresh cowhide is pretreated by dehairing, defatting, etc. and then chopped, and then placed in a 0.5 mol / L acetic acid solution containing 3% pepsin (EC 1:10000) for extraction. Centrifuge for 10 minutes using a high-speed refrigerated centrifuge to separate the supernatant. The precipitate obtained after sodium chloride salting out and centrifugation is redissolved in a 0.5 mol / L acetic acid solution. The dissolved collagen is dialyzed against a 0.1 mol / L acetic acid solution for three days, with the dialysate changed every 12 hours. Finally, the collagen solution is freeze-dried, and the collagen sponge obtained after freeze-drying is placed in a desiccator for storage.

[0054] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0055] The following examples were prepared using conventional instruments and equipment in the art. Experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or according to the conditions recommended by the manufacturer. The various raw materials used in the following examples, unless otherwise specified, were conventional commercially available products, with specifications conventional in the art. In the present specification and the following examples, unless otherwise specified, "%" indicates percentage by weight, "part" indicates parts by weight, and "ratio" indicates weight ratio.

[0056] Comparative Example 1

[0057] 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.

[0058] Example 1:

[0059] Collagen sponge and carboxymethyl chitosan were dissolved in a sodium salt solution of 10 mmol / L sodium dihydrogen phosphate / sodium dihydrogen phosphate and 125 mmol / L sodium chloride, respectively, to prepare collagen and carboxymethyl chitosan solutions. The collagen solution had a mass concentration of 1% and the carboxymethyl chitosan solution had a mass concentration of 1.5%. The volumes of the collagen and carboxymethyl chitosan solutions were equal, and the pH of each solution was controlled at 7.2 and the temperature was maintained at 4°C during the dissolution process. The carboxymethyl chitosan solution was slowly added dropwise to the stirring collagen solution at 4°C and stirred for 16 hours to prepare a collagen-carboxymethyl chitosan composite solution. Subsequently, the composite solution was allowed to self-assemble at 37°C for 3 hours to produce a collagen-carboxymethyl chitosan composite hydrogel. Trisodium citrate and zirconium sulfate tetrahydrate were dissolved in deionized water to prepare a zirconium cross-linking solution with a mass concentration of 3% zirconium sulfate tetrahydrate. The mass ratio of trisodium citrate to zirconium sulfate tetrahydrate was 1:1. The composite hydrogel was placed in a zirconium cross-linking solution and fixed with zirconium at 35°C for 72 hours, and the volume ratio of the collagen-carboxymethyl chitosan composite hydrogel to the zirconium cross-linking solution was 1:5; finally, the zirconium-fixed composite hydrogel was placed in a 3% titanyl sulfate solution and mineralized at 37°C for 48 hours to obtain a high-strength titanium mineralized collagen hydrogel, and the volume ratio of the zirconium cross-linked collagen-carboxymethyl chitosan composite hydrogel to the titanyl sulfate solution was 1:6.

[0060] The storage modulus of the titanium mineralized collagen hydrogel suitable for bone repair material prepared in this example is 120.0 kPa.

[0061] Example 2:

[0062] Collagen sponge and carboxymethyl chitosan were dissolved in 150 mmol / L sodium chloride solution to prepare collagen and carboxymethyl chitosan solutions, respectively. The collagen solution had a mass concentration of 2% and the carboxymethyl chitosan solution had a mass concentration of 8%. The volumes of the collagen and carboxymethyl chitosan solutions were equal, and the pH of each solution was controlled at 8 and the temperature at 10°C during the dissolution process. The carboxymethyl chitosan solution was slowly added dropwise to the stirring collagen solution at 10°C and stirred for 24 hours to prepare a collagen-carboxymethyl chitosan composite solution. Subsequently, the composite solution was allowed to self-assemble at 37°C for 3 hours to produce a collagen-carboxymethyl chitosan composite hydrogel. Trisodium citrate and zirconium sulfate tetrahydrate were dissolved in deionized water to prepare a zirconium cross-linking solution with a mass concentration of 5% zirconium sulfate tetrahydrate. The mass ratio of trisodium citrate to zirconium sulfate tetrahydrate was 1:10. The composite hydrogel was placed in a zirconium cross-linking solution and fixed with zirconium at 40°C for 96 hours, and the volume ratio of the collagen-carboxymethyl chitosan composite hydrogel to the zirconium cross-linking solution was 1:5; finally, the zirconium-fixed composite hydrogel was placed in a 20% titanyl sulfate solution and mineralized at 40°C for 72 hours to obtain a high-strength titanium mineralized collagen hydrogel, and the volume ratio of the zirconium cross-linked collagen-carboxymethyl chitosan composite hydrogel to the titanyl sulfate solution was 1:10.

[0063] The storage modulus of the titanium mineralized collagen hydrogel suitable for bone repair material prepared in this example is 264.5 kPa.

[0064] Example 3:

[0065] Collagen sponge and carboxymethyl chitosan were dissolved in 200 mmol / L sodium hydrogen phosphate solution to prepare collagen and carboxymethyl chitosan solutions, respectively. The collagen solution had a mass concentration of 0.4% and the carboxymethyl chitosan solution had a mass concentration of 0.1%. The volumes of the collagen and carboxymethyl chitosan solutions were equal, and the pH of each solution was controlled at 7 and the temperature at 2°C during the dissolution process. The carboxymethyl chitosan solution was slowly added dropwise to the stirring collagen solution at 2°C and stirred for 12 hours to prepare a collagen-carboxymethyl chitosan composite solution. Subsequently, the composite solution was allowed to self-assemble at 30°C for 3 hours to produce a collagen-carboxymethyl chitosan composite hydrogel. Trisodium citrate and zirconium sulfate tetrahydrate were dissolved in deionized water to prepare a zirconium cross-linking solution with a concentration of 0.1% zirconium sulfate tetrahydrate. The mass ratio of trisodium citrate to zirconium sulfate tetrahydrate was 1:1. The composite hydrogel was placed in a zirconium cross-linking solution and fixed with zirconium at 25°C for 48 hours, and the volume ratio of the collagen-carboxymethyl chitosan composite hydrogel to the zirconium cross-linking solution was 1:1; finally, the zirconium-fixed composite hydrogel was placed in a 0.1% titanyl sulfate solution and mineralized at 25°C for 12 hours to obtain a high-strength titanium mineralized collagen hydrogel, and the volume ratio of the zirconium cross-linked collagen-carboxymethyl chitosan composite hydrogel to the titanyl sulfate solution was 1:2.

[0066] The storage modulus of the titanium mineralized collagen hydrogel suitable for bone repair material prepared in this example is 17.8 kPa.

[0067] Example 4:

[0068] Collagen sponge and carboxymethyl chitosan were dissolved in 100 mmol / L sodium chloride solution to prepare collagen and carboxymethyl chitosan solutions, respectively. The collagen solution had a mass concentration of 1.2% and the carboxymethyl chitosan solution had a mass concentration of 2.4%. The volumes of the collagen and carboxymethyl chitosan solutions were equal, and the pH of each solution was controlled at 7.4 and the temperature at 7°C during the dissolution process. The carboxymethyl chitosan solution was slowly added dropwise to the stirring collagen solution at 7°C and stirred for 18 hours to prepare a collagen-carboxymethyl chitosan composite solution. Subsequently, the composite solution was allowed to self-assemble at 32°C for 3 hours to produce a collagen-carboxymethyl chitosan composite hydrogel. Trisodium citrate and zirconium sulfate tetrahydrate were dissolved in deionized water to prepare a zirconium cross-linking solution with a concentration of 1% zirconium sulfate tetrahydrate. The mass ratio of trisodium citrate to zirconium sulfate tetrahydrate was 1:2.5. The composite hydrogel was placed in a zirconium cross-linking solution and fixed with zirconium at 30°C for 84 hours, and the volume ratio of the collagen-carboxymethyl chitosan composite hydrogel to the zirconium cross-linking solution was 1:4; finally, the zirconium-fixed composite hydrogel was placed in a 10% titanyl sulfate solution and mineralized at 30°C for 48 hours to obtain a high-strength titanium mineralized collagen hydrogel, and the volume ratio of the zirconium cross-linked collagen-carboxymethyl chitosan composite hydrogel to the titanyl sulfate solution was 1:4.

[0069] The storage modulus of the titanium mineralized collagen hydrogel suitable for bone repair material prepared in this example is 137.9 kPa.

[0070] Example 5:

[0071] Collagen sponge and carboxymethyl chitosan were dissolved in 160 mmol / L sodium dihydrogen phosphate solution to prepare collagen and carboxymethyl chitosan solutions, respectively. The collagen solution had a mass concentration of 1.5% and the carboxymethyl chitosan solution had a mass concentration of 6%. The volumes of the collagen and carboxymethyl chitosan solutions were equal, and the pH of each solution was controlled at 7.6 and the temperature at 8°C during the dissolution process. The carboxymethyl chitosan solution was slowly added dropwise to the stirring collagen solution at 8°C and stirred for 20 hours to prepare a collagen-carboxymethyl chitosan composite solution. Subsequently, the composite solution was allowed to self-assemble at 35°C for 3 hours to produce a collagen-carboxymethyl chitosan composite hydrogel. Trisodium citrate and zirconium sulfate tetrahydrate were dissolved in deionized water to prepare a zirconium cross-linking solution with a concentration of 3% zirconium sulfate tetrahydrate. The mass ratio of trisodium citrate to zirconium sulfate tetrahydrate was 1:3. The composite hydrogel was placed in a zirconium cross-linking solution and fixed with zirconium at 35°C for 60 hours, and the volume ratio of the collagen-carboxymethyl chitosan composite hydrogel to the zirconium cross-linking solution was 1:7; finally, the zirconium-fixed composite hydrogel was placed in a 15% titanyl sulfate solution and mineralized at 35°C for 60 hours to obtain a high-strength titanium mineralized collagen hydrogel, and the volume ratio of the zirconium cross-linked collagen-carboxymethyl chitosan composite hydrogel to the titanyl sulfate solution was 1:3.

[0072] The storage modulus of the titanium mineralized collagen hydrogel suitable for bone repair material prepared in this example is 151.5 kPa.

[0073] Example 6

[0074] Collagen sponge and carboxymethyl chitosan were dissolved in 125 mmol / L sodium dihydrogen phosphate solution to prepare collagen and carboxymethyl chitosan solutions, respectively. The collagen solution had a mass concentration of 1.8% and the carboxymethyl chitosan solution had a mass concentration of 5.2%. The volumes of the collagen and carboxymethyl chitosan solutions were equal, and the pH of each solution was controlled at 7 and the temperature at 5°C during the dissolution process. The carboxymethyl chitosan solution was slowly added dropwise to the stirring collagen solution at 5°C and stirred for 20 hours to prepare a collagen-carboxymethyl chitosan composite solution. Subsequently, the composite solution was allowed to self-assemble at 35°C for 3 hours to produce a collagen-carboxymethyl chitosan composite hydrogel. Trisodium citrate and zirconium sulfate tetrahydrate were dissolved in deionized water to prepare a zirconium cross-linking solution with a mass concentration of 3% zirconium sulfate tetrahydrate. The mass ratio of trisodium citrate to zirconium sulfate tetrahydrate was 1:3.5. The composite hydrogel was placed in a zirconium cross-linking solution and fixed with zirconium at 35°C for 65 hours, and the volume ratio of the collagen-carboxymethyl chitosan composite hydrogel to the zirconium cross-linking solution was 1:7; finally, the zirconium-fixed composite hydrogel was placed in an 8% titanyl sulfate solution and mineralized at 30°C for 70 hours to obtain a high-strength titanium mineralized collagen hydrogel, and the volume ratio of the zirconium cross-linked collagen-carboxymethyl chitosan composite hydrogel to the titanyl sulfate solution was 1:7.5.

[0075] The storage modulus of the titanium mineralized collagen hydrogel suitable for bone repair material prepared in this embodiment is 233.7 kPa.

[0076] Example 7

[0077] Collagen sponge and carboxymethyl chitosan were dissolved in 120 mmol / L sodium hydrogen phosphate solution to prepare collagen and carboxymethyl chitosan solutions, respectively. The collagen solution had a mass concentration of 1% and the carboxymethyl chitosan solution had a mass concentration of 1%. The volumes of the collagen and carboxymethyl chitosan solutions were equal, and the pH of each solution was controlled at 7.1 and the temperature was maintained at 2°C during the dissolution process. The carboxymethyl chitosan solution was slowly added dropwise to the stirring collagen solution at 2°C and stirred for 16 hours to prepare a collagen-carboxymethyl chitosan composite solution. Subsequently, the composite solution was allowed to self-assemble at 36°C for 3 hours to produce a collagen-carboxymethyl chitosan composite hydrogel. Trisodium citrate and zirconium sulfate tetrahydrate were dissolved in deionized water to prepare a zirconium cross-linking solution with a concentration of 0.5% zirconium sulfate tetrahydrate. The mass ratio of trisodium citrate to zirconium sulfate tetrahydrate was 1:2. The composite hydrogel was placed in a zirconium cross-linking solution and fixed with zirconium at 30°C for 60 hours, and the volume ratio of the collagen-carboxymethyl chitosan composite hydrogel to the zirconium cross-linking solution was 1:5; finally, the zirconium-fixed composite hydrogel was placed in a 0.5% titanyl sulfate solution and mineralized at 30°C for 48 hours to obtain a high-strength titanium mineralized collagen hydrogel, and the volume ratio of the zirconium cross-linked collagen-carboxymethyl chitosan composite hydrogel to the titanyl sulfate solution was 1:5.

[0078] The storage modulus of the titanium mineralized collagen hydrogel suitable for bone repair material prepared in this example is 73.4 kPa.

[0079] Example 8

[0080] Collagen sponge and carboxymethyl chitosan were dissolved in a sodium salt solution of 20 mmol / L sodium dihydrogen phosphate / sodium dihydrogen phosphate and 100 mmol / L sodium chloride to prepare collagen and carboxymethyl chitosan solutions, respectively. The collagen solution had a mass concentration of 0.6% and the carboxymethyl chitosan solution had a mass concentration of 0.3%. The volumes of the collagen and carboxymethyl chitosan solutions were equal, and the pH of each solution was controlled at 7.5 and the temperature was maintained at 5°C during the dissolution process. The carboxymethyl chitosan solution was slowly added dropwise to the stirring collagen solution at 5°C and stirred for 24 hours to prepare a collagen-carboxymethyl chitosan composite solution. Subsequently, the composite solution was allowed to self-assemble at 37°C for 3 hours to produce a collagen-carboxymethyl chitosan composite hydrogel. Trisodium citrate and zirconium sulfate tetrahydrate were dissolved in deionized water to prepare a zirconium cross-linking solution with a concentration of 1.2% zirconium sulfate tetrahydrate. The mass ratio of trisodium citrate to zirconium sulfate tetrahydrate was 1:2.5. The composite hydrogel was placed in a zirconium cross-linking solution and fixed with zirconium at 35°C for 70 hours, and the volume ratio of the collagen-carboxymethyl chitosan composite hydrogel to the zirconium cross-linking solution was 1:7.5; finally, the zirconium-fixed composite hydrogel was placed in a 2.5% titanyl sulfate solution and mineralized at 35°C for 55 hours to obtain a high-strength titanium mineralized collagen hydrogel, and the volume ratio of the zirconium cross-linked collagen-carboxymethyl chitosan composite hydrogel to the titanyl sulfate solution was 1:8.

[0081] The storage modulus of the titanium mineralized collagen hydrogel suitable for bone repair material prepared in this example is 56.2 kPa.

[0082] Example 9

[0083] Collagen sponge and carboxymethyl chitosan were dissolved in a 160 mmol / L sodium chloride sodium salt solution to prepare a collagen solution and a carboxymethyl chitosan solution, respectively. The collagen solution had a mass concentration of 0.8% and the carboxymethyl chitosan solution had a mass concentration of 1.5%. The volumes of the collagen and carboxymethyl chitosan solutions were equal, and the pH and temperature of each solution were controlled at 8°C during the dissolution process. The carboxymethyl chitosan solution was slowly added dropwise to the stirring collagen solution at 8°C and stirred for 24 hours to prepare a collagen-carboxymethyl chitosan composite solution. Subsequently, the composite solution was allowed to self-assemble at 34°C for 3 hours to produce a collagen-carboxymethyl chitosan composite hydrogel. Trisodium citrate and zirconium sulfate tetrahydrate were dissolved in deionized water to prepare a zirconium cross-linking solution with a mass concentration of 2% zirconium sulfate tetrahydrate. The mass ratio of trisodium citrate to zirconium sulfate tetrahydrate was 1:4.5. The composite hydrogel was placed in a zirconium cross-linking solution and fixed with zirconium at 40°C for 80 hours, and the volume ratio of the collagen-carboxymethyl chitosan composite hydrogel to the zirconium cross-linking solution was 1:10; finally, the zirconium-fixed composite hydrogel was placed in a 5% titanyl sulfate solution and mineralized at 37°C for 60 hours to obtain a high-strength titanium mineralized collagen hydrogel, and the volume ratio of the zirconium cross-linked collagen-carboxymethyl chitosan composite hydrogel to the titanyl sulfate solution was 1:3.5.

[0084] The storage modulus of the titanium mineralized collagen hydrogel suitable for bone repair material prepared in this embodiment is 102.6 kPa.

[0085] Example 10

[0086] Collagen sponge and carboxymethyl chitosan were dissolved in 100 mmol / L sodium dihydrogen phosphate / sodium dihydrogen phosphate solutions to prepare collagen and carboxymethyl chitosan solutions, respectively. The collagen solution had a mass concentration of 1% and the carboxymethyl chitosan solution had a mass concentration of 2%. The volumes of the collagen and carboxymethyl chitosan solutions were equal, and the pH of each solution was controlled at 8 and the temperature at 10°C during the dissolution process. The carboxymethyl chitosan solution was slowly added dropwise to the stirring collagen solution at 10°C and stirred for 19 hours to prepare a collagen-carboxymethyl chitosan composite solution. Subsequently, the composite solution was allowed to self-assemble at 33°C for 3 hours to produce a collagen-carboxymethyl chitosan composite hydrogel. Trisodium citrate and zirconium sulfate tetrahydrate were dissolved in deionized water to prepare a zirconium cross-linking solution with a zirconium sulfate tetrahydrate concentration of 2.5%. The mass ratio of trisodium citrate to zirconium sulfate tetrahydrate was 1:5. The composite hydrogel was placed in a zirconium cross-linking solution and fixed with zirconium at 36°C for 96 hours, and the volume ratio of the collagen-carboxymethyl chitosan composite hydrogel to the zirconium cross-linking solution was 1:4; finally, the zirconium-fixed composite hydrogel was placed in a 10% titanyl sulfate solution and mineralized at 40°C for 72 hours to obtain a high-strength titanium mineralized collagen hydrogel, and the volume ratio of the zirconium cross-linked collagen-carboxymethyl chitosan composite hydrogel to the titanyl sulfate solution was 1:6.5.

[0087] The storage modulus of the titanium mineralized collagen hydrogel suitable for bone repair material prepared in this example is 91.4 kPa.

[0088] Figure 2 The following are scanning electron micrographs of pure collagen (Comparative Example 1) and titanium-mineralized collagen hydrogel (Example 1). It can be observed that the fiber surface of the pure collagen hydrogel is smooth, while the fiber surface of the mineralized collagen hydrogel is coated with a large amount of spherical TiO2.

[0089] Figure 3 The storage modulus of pure collagen (Comparative Example 1) and titanium-mineralized collagen hydrogel (Example 1) is about 1320 times greater than that of unmineralized pure collagen hydrogel (90.8 Pa), indicating that the introduction of TiO2 imparts excellent mechanical properties.

[0090] The storage modulus test process in this application is specifically as follows:

[0091] The hydrogel was placed in the rheometer's plate fixture (20 mm diameter, 1.0 mm spacing between plates) and subjected to a stress-strain sweep. The hydrogel's linear viscoelastic region was determined, and an appropriate strain level (1%) was selected within this region. A dynamic frequency sweep was then performed in strain-controlled mode to determine the storage modulus. The test temperature was 25 ± 0.1°C, and the frequency range was 0.01–10 Hz.

[0092] Storage modulus, also known as elastic modulus, is an indicator that measures the ease with which a material undergoes elastic deformation. The larger its value, the greater the stress required to cause a certain elastic deformation of the material, that is, the greater the material stiffness, that is, the smaller the elastic deformation under a certain stress. Generally represented by G', the unit of storage modulus is Pascal (Pa), kilopascal (kPa) or megapascal (MPa). The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be considered as the scope of protection of the present invention.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing titanium mineralized collagen hydrogel suitable for bone repair material, characterized in that: The following steps are involved: S1: Dissolving collagen sponge and carboxymethyl chitosan in sodium salt solution to prepare collagen solution and carboxymethyl chitosan solution respectively; S2: adding the carboxymethyl chitosan solution dropwise to the collagen solution, stirring to react, and obtaining a collagen-carboxymethyl chitosan composite solution; S3: allowing the collagen-carboxymethyl chitosan composite solution to self-assemble into fibers to prepare a collagen-carboxymethyl chitosan composite hydrogel; S4: placing the collagen-carboxymethyl chitosan composite hydrogel in a zirconium cross-linking solution to react, thereby obtaining a zirconium-cross-linked collagen-carboxymethyl chitosan composite hydrogel; S5: placing the zirconium-crosslinked collagen-carboxymethyl chitosan composite hydrogel in a titanyl sulfate solution to carry out a mineralization reaction, thereby obtaining the titanium-mineralized collagen hydrogel suitable for bone repair material.

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

3. The method for preparing a titanium mineralized collagen hydrogel suitable for bone repair material according to claim 1, characterized in that: 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 to be 7-8, the reaction temperature is 2-10°C, and the stirring time is 12-24h.

4. The method for preparing a titanium mineralized collagen hydrogel suitable for bone repair material according to claim 1, characterized in that: In step S3, specifically, the collagen-carboxymethyl chitosan composite solution is placed at 30-37° C. for self-assembly to obtain the collagen-carboxymethyl chitosan composite hydrogel.

5. The method for preparing a titanium mineralized collagen hydrogel suitable for bone repair material according to claim 1, characterized in that: 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); and the mass concentration of zirconium sulfate tetrahydrate in the zirconium cross-linking liquid is 0.1%-5%.

6. The method for preparing a titanium mineralized collagen hydrogel suitable for bone repair material according to claim 1, characterized in that: In step S4, the volume ratio of the collagen-carboxymethyl chitosan composite hydrogel to the zirconium cross-linking liquid is 1:(1-10); the reaction temperature in step S4 is 25-40° C., and the reaction time is 48-96 hours.

7. The method for preparing a titanium mineralized collagen hydrogel suitable for bone repair material according to claim 1, characterized in that: In step S5, the mass concentration of the titanyl 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 to 10).

8. The method for preparing a titanium mineralized collagen hydrogel suitable for bone repair material according to claim 1, characterized in that: In step S5, the mineralization process is specifically as follows: after mixing the zirconium-crosslinked collagen-carboxymethyl chitosan composite hydrogel with the titanium sulfate solution, the mixture is placed at 25-40° C. for mineralization for 24-72 hours to obtain the titanium mineralized collagen hydrogel suitable for bone repair material.

9. A titanium mineralized collagen hydrogel suitable for bone repair material, characterized in that: The titanium mineralized collagen hydrogel suitable for bone repair material is prepared by the method according to any one of claims 1 to 8, and has a storage modulus of 17.8 to 264.5 kPa.

10. A bone repair material, characterized in that: The invention comprises the titanium mineralized collagen hydrogel suitable for bone repair material as described in claim 9.

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