A hydroxyapatite-collagen material and its preparation method and application

Abstract

The present invention provides a hydroxyapatite-collagen material, its preparation method, and application. The hydroxyapatite-collagen material comprises collagen fibers, alginic acid, and hydroxyapatite. The alginic acid is grafted onto the collagen fibers by forming amide bonds with the collagen fibers. The hydroxyapatite is grafted onto the collagen fibers by crosslinking the alginic acid with calcium ions. The present invention achieves crosslinking of the collagen fibers through self-crosslinking, significantly reducing toxicity and increasing collagen content. The resulting hydroxyapatite-collagen material exhibits excellent mechanical properties.

Description

Technical Field

[0001] The present invention relates to the technical field of bone filling materials, in particular to a hydroxyapatite-collagen material and a preparation method and application thereof, and particularly to a self-crosslinked hydroxyapatite / collagen bone filling material and a preparation method and application thereof. Background Art

[0002] Natural bone is a complex composed of regularly arranged inorganic minerals and biomacromolecules. The inorganic material is primarily hydroxyapatite, while the biomacromolecules primarily include collagen, both of which are uniformly mixed and orderly bound together. Therefore, the key to artificial bone material research is to obtain an artificial bone material that is identical to natural bone tissue. This is because if the artificial bone has a similar size to that of the microcrystalline hydroxyapatite nucleated on the collagen fibers of natural bone tissue, it will be easier for human cells and macromolecules to recognize it, thereby improving the material's bioactivity and biocompatibility.

[0003] The cross-linking method of collagen plays a significant role in the performance of artificial bone materials. Common cross-linking methods include three types: physical cross-linking, chemical cross-linking and biological cross-linking. Physical cross-linking includes high-temperature dehydration heat treatment (DHT) and ultraviolet radiation cross-linking. DHT is a process in which collagen fibers are dehydrated at high temperatures above 100°C in a vacuum environment to form cross-links between collagen fibers. During this process, the amino and carboxyl groups in the collagen fibers undergo esterification reactions at high temperatures, thereby producing cross-linked bonds. Ultraviolet radiation cross-linking is the process in which some OH· free radicals are generated in the solution under ultraviolet light. The OH· free radicals attack the peptide backbone to produce -NH-C*-CO-, causing the collagen fibers to cross-link:

[0004] Chemical cross-linking is the process of chemical reactions between collagens to form new covalent bonds and form a network structure. There are many types of reactions, and the following are commonly used:

[0005] (1) Schiff base reaction

[0006] Compounds containing carbonyl groups, such as aldehydes and ketones, can undergo addition reactions with amines to form α-hydroxy intermediates, which then dehydrate to form Schiff base products. This reaction can be regulated by pH, with higher yields achieved at lower pH levels. However, the double bonds carried by carbonyl groups, such as aldehydes, ketones, and glyoxal, are highly reactive. If the cross-linking reaction is incomplete or the incompletely reacted double bonds cannot be removed, they may cause adverse reactions such as erythema and edema upon entry into the body.

[0007] For example, CN102406965A discloses an injectable gel material for treating bone defects and its preparation method. The injectable gel material is obtained by selectively oxidizing the ortho-hydroxyl groups in sodium alginate molecules to aldehyde groups, which are then mixed with nanohydroxyapatite and collagen. The aldehyde groups in the sodium alginate molecules react with the amino groups in the collagen molecules to form Schiff's base, thereby chemically cross-linking the sodium alginate and collagen. However, as mentioned above, if the cross-linking reaction is incomplete or the incompletely reacted double bonds cannot be removed, the material may cause adverse reactions such as erythema and edema after entering the body. In addition, the material is an injectable gel with low mechanical strength, making it unsuitable for use in the field of bone filling materials.

[0008] (2) Carbodiimides

[0009] EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) is the most commonly used type of carbodiimide cross-linking agent. First, EDC reacts with collagen to form active O-urea, and then combines with amino groups to form amide bonds, releasing isourea. This isourea product has good water solubility. Although it has weak toxicity to cells, it can be removed from the system through simple treatment methods such as washing and dialysis. The advantage of EDC is that it is a zero-length cross-linking agent. When two molecules are cross-linked together through EDC, no other substances are introduced in the middle to form a "bridge"-like structure, but the two molecules react directly. In order to improve the efficiency of the coupling reaction, N-hydroxysuccinimide (NHS) is usually added in a certain proportion. The combination of the two greatly improves the stability of the intermediate product generated by the reaction, thereby effectively improving the degree of cross-linking.

[0010] Because collagen exhibits certain toxicity after chemical cross-linking with EDC, residual chemical cross-linkers can be removed through washing. For example, CN114404668A discloses a method for producing an injectable collagen filler free of cross-linker residue. Using EDC to cross-link collagen uses a low concentration of cross-linker, is simple, and can be performed easily. The degree of cross-linking can be increased through secondary cross-linking. After the cross-linking reaction is complete, the EDC can be essentially completely removed with a simple phosphate buffer wash.

[0011] (3) Polyepoxides

[0012] Among polyepoxy crosslinkers, 1,4-butanediol diglycidyl ether (BDDE) is the most commonly used. Its crosslinking mechanism with collagen depends largely on the reactivity of the epoxy groups present at the ends of the molecules. Furthermore, crosslinking reactivity is also dependent on pH and temperature conditions. Under alkaline pH conditions, the epoxide ring opens and reacts with the amine groups to form strong ether bonds, connecting the molecules. However, the crosslinks formed by polyepoxides are irreversible and do not degrade in the body, making them difficult to integrate with tissues.

[0013] (4) Glutaraldehyde (GTA)

[0014] GTA is a bifunctional cross-linker that forms covalent bonds between the amine groups of lysine or hydroxylysine residues in polypeptide chains and the aldehyde groups of GTA, thereby enhancing the degradation resistance of proteins. Despite being the most extensively studied cross-linker, GTA's biocompatibility remains a concern. Its advantages include low cost, easy availability, rapid reaction, and stability. However, GTA is cytotoxic and harmful to tissues, as it can induce adverse immune responses.

[0015] Biocrosslinking uses enzymes, biopolysaccharides, and other materials as crosslinking agents to crosslink collagen fibers. A common biocrosslinking agent is transglutaminase (TGase). TGase catalyzes the amidotransfer reaction between the γ-amide group of glutamine residues in proteins and the ε-amino group of lysine, forming an ε-(γ-glutamine)-lysine heteropeptide bond, thereby crosslinking the collagen fibers.

[0016] Each of these cross-linking methods has its advantages and disadvantages. Physical cross-linking offers the greatest advantage of being non-toxic, but the high temperatures and irradiation involved in the cross-linking process can easily cause structural changes in the collagen. Chemical cross-linking, while adding a cross-linking agent, results in a stable cross-linked network with improved performance, but the cross-linking agents used are toxic. While biological cross-linking can largely offset the shortcomings of physical and chemical cross-linking methods, it comes at a high cost.

[0017] Existing solutions include washing away residual chemical crosslinkers or replacing them with less toxic crosslinkers. However, these solutions only reduce toxicity by changing or cleaning the crosslinker. However, some crosslinkers remain, and the resulting collagen network still retains some toxicity, effectively treating the symptoms rather than the underlying cause.

[0018] In view of this, the present invention is proposed. Summary of the Invention

[0019] One of the objectives of the present invention is to provide a hydroxyapatite-collagen material. The hydroxyapatite-collagen material comprises collagen fibers, alginic acid, and hydroxyapatite. The alginic acid is grafted onto the collagen fibers by forming amide bonds with the collagen fibers. The hydroxyapatite is grafted onto the collagen fibers by crosslinking the alginic acid with calcium ions. The crosslinking of the collagen fibers is achieved through a non-toxic self-crosslinking method, while exhibiting excellent mechanical strength.

[0020] A second object of the present invention is to provide a method for preparing the hydroxyapatite-collagen material. The preparation method utilizes the cross-linking reaction between alginate and calcium ions to graft alginate onto collagen fibers, and then utilizes the calcium ions in hydroxyapatite to achieve a cross-linking reaction between alginate and calcium ions; the cross-linking of collagen fibers is achieved in a self-cross-linking manner to form a cross-linked network, thereby avoiding the generation of toxicity and at the same time avoiding the damage to the collagen structure caused by high temperature.

[0021] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0022] In a first aspect, the present invention provides a hydroxyapatite-collagen material, wherein the hydroxyapatite-collagen material comprises: collagen fibers, alginic acid and hydroxyapatite;

[0023] The alginic acid is grafted onto the collagen fiber by forming an amide bond with the collagen fiber;

[0024] The hydroxyapatite is grafted onto the collagen fiber through the cross-linking of calcium ions and alginic acid.

[0025] In the present invention, alginic acid is introduced into collagen fibers through an amidation reaction. Alginic acid is a polysaccharide with many negative charges, while calcium ions are cations with two positive charges. When alginic acid and calcium ions meet, they form a stable cross-linked structure. The cross-linking mechanism is through electrostatic interaction. The negative charges in the alginic acid molecules attract the positive charges in the calcium ions, allowing the collagen fibers to undergo a cross-linking reaction with the calcium ions in hydroxyapatite, forming a cross-linked network. This avoids toxicity and simultaneously prevents high temperature damage to the collagen structure.

[0026] Furthermore, because alginate is grafted onto collagen fibers by forming amide bonds, it aids in the cross-linking of hydroxyapatite, further enhancing the toughness of the collagen fibers and the strength of the hydroxyapatite. This results in superior mechanical properties for the hydroxyapatite-collagen material prepared by the present invention. The composition and microstructure of this scaffold material are similar to those of natural bone, further facilitating the differentiation and osteogenesis of human bone marrow mesenchymal stem cells.

[0027] Preferably, the mass ratio of the collagen fibers, alginate and hydroxyapatite is 100:(0.3–0.5):(20-40);

[0028] Here, “0.3–0.5” can be, for example, 0.3, 0.35, 0.4, 0.45, 0.5, etc.;

[0029] Here, “20-40” can be, for example, 20, 25, 30, 35, 40, etc.

[0030] Preferably, the raw materials for preparing the hydroxyapatite-collagen material include the following components: collagen slurry, halogenation reagent, alginic acid, calcium chloride, and sodium phosphate.

[0031] Preferably, the molecular weight of the collagen is 10-200 kDa, for example, 10 kDa, 50 kDa, 100 kDa, 150 kDa, 200 kDa, etc., preferably 120-140 kDa.

[0032] Preferably, the concentration of the collagen slurry is 2-8 g / L, for example, it can be 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, etc.

[0033] Preferably, the halogenating agent is a phosphorus halide, preferably phosphorus pentachloride.

[0034] Preferably, the mass ratio of the collagen slurry, the halogenated reagent, and the alginic acid is 100:(3-6):(0.3-0.5);

[0035] Among them, "3-6" can be, for example, 3, 3.5, 4, 4.5, 5, 5.5, 6, etc.;

[0036] Here, “0.3-0.5” can be, for example, 0.3, 0.35, 0.4, 0.45, 0.5, etc.

[0037] Preferably, the molar ratio of calcium chloride to sodium phosphate is 1:(1.1-1.5), for example, it can be 1:1.1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, preferably 1:1.2.

[0038] Preferably, the raw materials for preparing the hydroxyapatite-collagen material further include an acidic pH regulator and / or an alkaline pH regulator.

[0039] Preferably, the acidic pH adjuster comprises acetic acid.

[0040] Preferably, the alkaline pH adjuster comprises an aqueous solution of NaOH.

[0041] Preferably, the mass percentage of the aqueous solution of NaOH is 1-20%, for example, it can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, etc.

[0042] In a second aspect, the present invention provides a method for preparing the hydroxyapatite-collagen material as described in the first aspect, the preparation method comprising the following steps:

[0043] Through the amidation reaction between the carboxyl groups in alginate and the amino groups in collagen fibers, alginate is grafted onto the collagen fibers to obtain alginate-collagen fibers;

[0044] Calcium ions and phosphate ions are reacted on the alginate-collagen fibers to form hydroxyapatite by an in-situ deposition method;

[0045] The hydroxyapatite-collagen material is obtained by cross-linking reaction between calcium ions in the hydroxyapatite and the alginate-collagen fibers.

[0046] The present invention first utilizes an amidation reaction to graft alginate onto collagen fibers, allowing the carboxyl groups in alginate to react with the amino groups in collagen. Calcium ions and phosphate ions react on the collagen fibers through in-situ deposition. Finally, continuous stirring is used to allow the added calcium ions to crosslink with the collagen fibers containing alginate, forming a crosslinked network. The crosslinking process eliminates the need for chemical crosslinking agents or high-temperature heat treatment, achieving maximum non-toxic crosslinking while preserving the collagen structure. Post-processing eliminates the need for washing and allows for direct drying to yield a pure finished product.

[0047] Preferably, the amidation reaction specifically comprises the following steps:

[0048] (a) mixing alginic acid and a halogenating agent to cause a halogenation reaction to obtain a halogenated product;

[0049] (b) reacting the halogenated product with collagen to obtain alginate-collagen fibers.

[0050] Preferably, in step (a), the temperature of the halogenation reaction is 10-30°C, for example, 10°C, 15°C, 20°C, 25°C, 30°C, etc., and the time of the halogenation reaction is 1-3h, for example, 1h, 1.5h, 1.8h, 2h, 2.2h, 2.5h, 3h, etc.

[0051] Preferably, in step (a), the halogenation reaction needs to be carried out at a pH of 8-9 (eg, 8, 8.2, 8.4, 8.6, 8.8, 9, etc.).

[0052] Preferably, in step (a), the pH of the halogenation reaction is adjusted to 8-9 (for example, 8, 8.2, 8.4, 8.6, 8.8, 9, etc.) using NaOH solution.

[0053] Preferably, in step (b), the reaction temperature is 40-60°C, for example, 40°C, 45°C, 50°C, 55°C, 60°C, etc., and the reaction time is 7-9h, for example, 7h, 7.5h, 8h, 8.5h, 9h, etc.

[0054] Preferably, before the reaction of the calcium ions and phosphate ions, the pH of the alginate-collagen fiber system needs to be adjusted to 6.8-7.2, for example, 6.8, 6.9, 7, 7.1, 7.2, etc.

[0055] Preferably, the in-situ deposition method specifically comprises the following steps: simultaneously adding calcium chloride solution and sodium phosphate solution to the alginate-collagen fiber system.

[0056] Preferably, the concentration of the calcium chloride solution is 0.18-0.22 mol / L, for example, it can be 0.18 mol / L, 0.19 mol / L, 0.20 mol / L, 0.21 mol / L, 0.22 mol / L, etc.

[0057] Preferably, the concentration of the sodium phosphate solution is 0.22-0.26 mol / L, for example, it can be 0.22 mol / L, 0.23 mol / L, 0.24 mol / L, 0.25 mol / L, 0.26 mol / L, etc.

[0058] Preferably, the calcium chloride solution is added at a rate of 1.7-1.9 g / min, for example, 1.7 g / min, 1.75 g / min, 1.8 g / min, 1.85 g / min, 1.9 g / min, etc.

[0059] Preferably, the dropping speed of the sodium phosphate solution is 1.5-1.7 g / min, for example, 1.5 g / min, 1.55 g / min, 1.6 g / min, 1.65 g / min, 1.7 g / min, etc.

[0060] Preferably, the difference in the dropping speed of the calcium chloride solution and the sodium phosphate solution is 0.1-0.3 g / min, for example, it can be 0.1 g / min, 0.15 g / min, 0.2 g / min, 0.25 g / min, 0.3 g / min, etc.

[0061] Preferably, the cross-linking reaction is carried out by stirring.

[0062] Preferably, the cross-linking reaction temperature is 80-120°C, for example, 80°C, 90°C, 100°C, 120°C, etc., and the cross-linking reaction time is 2-4h, for example, 2h, 2.5h, 3h, 3.5h, 4h, etc.

[0063] Preferably, the preparation method comprises the following steps:

[0064] (1) phosphorus pentachloride is added dropwise to an aqueous solution containing alginic acid, and the reaction is carried out at 10-30° C. for 1-3 hours, and a pH regulator is used during the reaction to maintain the pH of the reaction system between 8 and 9;

[0065] (2) adding alginic acid to the reaction solution obtained in step (1) and reacting at 40-60° C. for 7-9 hours;

[0066] (3) After adjusting the pH of the reaction solution obtained in step (2) to 6.8-7.2 using a pH regulator, calcium chloride solution is added dropwise at a rate of 1.7-1.9 g / min and sodium phosphate solution is added dropwise at a rate of 1.5-1.7 g / min, and the reaction is continued at 10-30° C. for 2-4 hours, and dried to obtain the hydroxyapatite-collagen material.

[0067] In a third aspect, the present invention provides a use of the hydroxyapatite-collagen material according to any one of claims 1 to 4 in preparing a bone filling material.

[0068] Compared with the prior art, the present invention has the following beneficial effects:

[0069] (1) The present invention provides a hydroxyapatite-collagen material, wherein alginate is grafted onto collagen fibers by forming amide bonds with the collagen fibers; hydroxyapatite is grafted onto the collagen fibers by cross-linking calcium ions with alginate, further improving the toughness of the collagen fibers and the strength of the hydroxyapatite, thereby making the mechanical properties of the hydroxyapatite-collagen material prepared by the present invention more excellent;

[0070] (2) The present invention first utilizes an amidation reaction to cause an amidation reaction between the carboxyl groups in alginate and the amino groups in collagen, thereby grafting alginate onto collagen fibers. Calcium ions and phosphate ions react on the collagen fibers through an in-situ deposition method. Finally, continuous stirring is used to cause a crosslinking reaction between the added calcium ions and the collagen fibers containing alginate, forming a crosslinked network. During the crosslinking process, no chemical crosslinking agent is required, and no high-temperature heat treatment is required. Non-toxic crosslinking is achieved to the greatest extent possible, while preserving the structure of the collagen. Post-processing does not require washing, and a pure finished product can be obtained by direct drying. DETAILED DESCRIPTION

[0071] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, the use of "or" means "and / or" unless otherwise stated. In addition, the use of the term "including" and other forms is non-limiting.

[0072] Generally, the nomenclature used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization as described herein and its technology are those well-known and commonly used in this area.Unless otherwise indicated, the methods and techniques of the present invention are generally according to those well-known in the art, and are carried out as described in various general and more specific references, which are cited and discussed throughout this specification.Enzymatic reactions and purification techniques are carried out according to the manufacturer's specifications, as commonly achieved in this area, or as described herein.The nomenclature used in conjunction with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry as described herein and its laboratory procedures and technology are those well-known and commonly used in this area.

[0073] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0074] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples were prepared according to existing methods or directly purchased from the market.

[0075] Example 1

[0076] This embodiment provides a hydroxyapatite-collagen material, which is prepared by the following method:

[0077] (1) 0.33 g of alginic acid was dissolved in 10 mL of a 10% aqueous NaOH solution, placed on a magnetic stirrer, and 3.6 mL of PCl5 was slowly added dropwise while stirring. The reaction was carried out at 20°C for 2 h, and the pH of the reaction system was controlled at 8.5 using a 10% aqueous NaOH solution during the reaction.

[0078] (2) Add 100 g of collagen slurry (molecular weight 130 kD, concentration 4 g / L) to the above reaction solution and react at 50°C for 8 h;

[0079] (3) Acetic acid was added to the above reaction solution to adjust the pH of the solution to 7. While stirring, 56 mL of calcium chloride and sodium phosphate solutions with concentrations of 0.2 mol / L and 0.24 mol / L were added dropwise at flow rates of 1.8 g / min and 1.6 g / min, respectively. The mixture was stirred at 30°C for 3 h. After the reaction was completed, the mixture was freeze-dried to obtain a hydroxyapatite-collagen material.

[0080] Example 2

[0081] This embodiment provides a hydroxyapatite-collagen material, which is prepared by the following method:

[0082] (1) 0.33 g of alginic acid was dissolved in 10 mL of a 10% aqueous NaOH solution, placed on a magnetic stirrer, and 3.6 mL of PCl5 was slowly added dropwise while stirring. The reaction was carried out at 20°C for 2 h, and the pH of the reaction system was controlled at 8.5 using a 10% aqueous NaOH solution during the reaction.

[0083] (2) Add 100 g of collagen slurry (molecular weight 130 kD, concentration 4 g / L) to the above reaction solution and react at 50°C for 8 h;

[0084] (3) Acetic acid was added to the above reaction solution to adjust the pH of the solution to 7. While stirring, 56 mL of calcium chloride and sodium phosphate solutions with concentrations of 0.2 mol / L and 0.24 mol / L were added dropwise at flow rates of 1.9 g / min and 1.5 g / min, respectively. The mixture was stirred at 30°C for 3 h. After the reaction was completed, the mixture was freeze-dried to obtain a hydroxyapatite-collagen material.

[0085] Example 3

[0086] This embodiment provides a hydroxyapatite-collagen material, which is prepared by the following method:

[0087] (1) 0.33 g of alginic acid was dissolved in 10 mL of a 10% aqueous NaOH solution, placed on a magnetic stirrer, and 3.6 mL of PCl5 was slowly added dropwise while stirring. The reaction was carried out at 20°C for 2 h, and the pH of the reaction system was controlled at 8.5 using a 10% aqueous NaOH solution during the reaction.

[0088] (2) Add 100 g of collagen slurry (molecular weight 130 kD, concentration 4 g / L) to the above reaction solution and react at 50°C for 8 h;

[0089] (3) Acetic acid was added to the above reaction solution to adjust the pH of the solution to 7. While stirring, 56 mL of calcium chloride and sodium phosphate solutions with concentrations of 0.2 mol / L and 0.24 mol / L were added dropwise at flow rates of 1.7 g / min and 1.7 g / min, respectively. The mixture was stirred at 30°C for 3 h. After the reaction was completed, the mixture was freeze-dried to obtain a hydroxyapatite-collagen material.

[0090] Example 4

[0091] This embodiment provides a hydroxyapatite-collagen material, which is prepared by the following method:

[0092] (1) 0.33 g of alginic acid was dissolved in 10 mL of a 10% aqueous NaOH solution, placed on a magnetic stirrer, and 3.6 mL of PCl5 was slowly added dropwise while stirring. The reaction was carried out at 20°C for 2 h, and the pH of the reaction system was controlled at 8.5 using a 10% aqueous NaOH solution during the reaction.

[0093] (2) Add 100 g of collagen slurry (molecular weight 130 kD, concentration 4 g / L) to the above reaction solution and react at 50°C for 8 h;

[0094] (3) Acetic acid was added to the above reaction solution to adjust the pH of the solution to 7. While stirring, 1.683 g of hydroxyapatite was directly added and stirred for 3 h. After the reaction was completed, lyophilization was performed to obtain a hydroxyapatite-collagen material.

[0095] Comparative Example 1

[0096] This comparative example provides a hydroxyapatite-collagen material, which is prepared by the following method:

[0097] Take 100 g of collagen slurry, adjust the pH of the solution to 7 with 1 mol / L sodium hydroxide, and add 56 mL of 0.2 mol / L and 0.24 mol / L calcium chloride and sodium phosphate solutions at the same time while stirring, at a flow rate of 1.8 g / min and 1.6 g / min, respectively. Stir for 3 hours. After the reaction is completed, freeze-dry and heat-treat.

[0098] Comparative Example 2

[0099] This comparative example provides a hydroxyapatite-collagen material, which is prepared by the following method:

[0100] (1) 0.33 g of hyaluronic acid was dissolved in 10 mL of a 10% aqueous NaOH solution, placed on a magnetic stirrer, and 3.6 mL of PCl5 was slowly added dropwise while stirring. The mixture was reacted at 20°C for 2 h, and the pH of the reaction system was controlled at 8.5 using a 10% aqueous NaOH solution during the reaction.

[0101] (2) Add 100 g of collagen slurry (molecular weight 130 kD, concentration 4 g / L) to the above reaction solution and react at 50°C for 8 h;

[0102] (3) Acetic acid was added to the above reaction solution to adjust the pH of the solution to 7. While stirring, 56 mL of calcium chloride and sodium phosphate solutions with concentrations of 0.2 mol / L and 0.24 mol / L were added dropwise at flow rates of 1.8 g / min and 1.6 g / min, respectively. The mixture was stirred at 30°C for 3 h. After the reaction was completed, the mixture was freeze-dried to obtain a hydroxyapatite-collagen material.

[0103] Test Example 1

[0104] Physical and chemical parameter testing

[0105] Test samples: hydroxyapatite-collagen materials prepared in Examples 1-4 and hydroxyapatite-collagen materials prepared in Comparative Examples 1-2;

[0106] Test method:

[0107] (1) Cross-linking degree: The cross-linking degree was determined by ultraviolet spectrophotometry;

[0108] (2) Porosity: Scanning electron microscopy results were analyzed, and porosity (%) = void area / total area;

[0109] The specific test results are shown in Table 1:

[0110] Table 1

[0111] Group Degree of crosslinking Porosity Example 1 92％ 96％ Example 2 86％ 92％ Example 3 82％ 87％ Example 4 88％ 93％ Comparative Example 1 53％ 85％ Comparative Example 2 42％ 78％

[0112] As shown in the test results in Table 1, the hydroxyapatite-collagen material prepared by the present invention has a crosslinking degree of 90%-94% and a porosity of 94%-98%. This indicates that the preparation process of the present invention enables collagen fibers to undergo a crosslinking reaction with the calcium ions in the hydroxyapatite, forming a tightly crosslinked network while avoiding toxicity and high-temperature damage to the collagen structure, resulting in a high collagen content.

[0113] Test Example 2

[0114] Mechanical properties testing

[0115] Test samples: hydroxyapatite-collagen materials prepared in Examples 1-4 and hydroxyapatite-collagen materials prepared in Comparative Examples 1-2;

[0116] Test method: Make the sample into a cylinder with a diameter of 10mm and a height of 20mm, measure its compressive force on a dynamometer, and calculate the compressive strength.

[0117] The specific test results are shown in Table 2:

[0118] Table 2

[0119]

[0120]

[0121] As shown in the test data in Table 2, the compressive strength of the hydroxyapatite-collagen material prepared by the present invention ranges from 4.8 MPa to 5.5 MPa, demonstrating that the collagen-hydroxyapatite artificial bone prepared by the present invention has excellent mechanical properties. Because alginate is grafted onto the collagen fibers by forming amide bonds with them, this aids in the cross-linking of hydroxyapatite, further improving the toughness of the collagen fibers and the strength of the hydroxyapatite. This results in even better mechanical properties for the hydroxyapatite-collagen material prepared by the present invention. The composition and microstructure of this scaffold material are similar to those of natural bone, making it more conducive to the differentiation and osteogenesis of human bone marrow mesenchymal stem cells.

[0122] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A hydroxyapatite-collagen material, characterized in that: The hydroxyapatite-collagen material comprises: collagen fiber, alginate and hydroxyapatite; The alginic acid is grafted onto the collagen fiber by forming an amide bond with the collagen fiber; The hydroxyapatite is grafted onto the collagen fiber through the cross-linking of calcium ions and alginate; The hydroxyapatite-collagen material is prepared by the following steps: Through the amidation reaction between the carboxyl groups in alginate and the amino groups in collagen fibers, alginate is grafted onto the collagen fibers to obtain alginate-collagen fibers; Calcium ions and phosphate ions are reacted on the alginate-collagen fibers to form hydroxyapatite by an in-situ deposition method; The hydroxyapatite-collagen material is obtained by cross-linking the calcium ions in the hydroxyapatite with the alginate-collagen fibers; Wherein, the amidation reaction specifically comprises the following steps: (a) mixing alginic acid and a halogenating agent to cause a halogenation reaction to obtain a halogenated product; (b) The halogenated product is reacted with collagen to obtain alginate-collagen fibers.

2. The hydroxyapatite-collagen material according to claim 1, characterized in that The mass ratio of the collagen fiber, alginate and hydroxyapatite is 100:(0.3–0.5):(20-40).

3. The hydroxyapatite-collagen material according to claim 1, characterized in that The raw materials for preparing the hydroxyapatite-collagen material include the following components: collagen slurry, halogenation reagent, alginic acid, calcium chloride and sodium phosphate.

4. The hydroxyapatite-collagen material according to claim 3, characterized in that The molecular weight of the collagen is 10-200 kDa.

5. The hydroxyapatite-collagen material according to claim 4, characterized in that The molecular weight of the collagen is 120-140 kDa.

6. The hydroxyapatite-collagen material according to claim 3, characterized in that The concentration of the collagen slurry is 2-8 g / L.

7. The hydroxyapatite-collagen material according to claim 3, characterized in that The halogenating agent is a phosphorus halide.

8. The hydroxyapatite-collagen material according to claim 7, characterized in that The halogenating agent is phosphorus pentachloride.

9. The hydroxyapatite-collagen material according to claim 3, characterized in that The mass ratio of the collagen slurry, the halogenation reagent and the alginic acid is 100:(3-6):(0.3-0.5).

10. The hydroxyapatite-collagen material according to claim 3, characterized in that The molar ratio of the calcium chloride to the sodium phosphate is 1:(1.1-1.5).

11. The hydroxyapatite-collagen material according to claim 10, characterized in that The molar ratio of the calcium chloride to the sodium phosphate is 1:1.

2.

12. The hydroxyapatite-collagen material according to claim 3, characterized in that The raw materials for preparing the hydroxyapatite-collagen material also include an acidic pH regulator and / or an alkaline pH regulator.

13. The hydroxyapatite-collagen material according to claim 12, characterized in that The acidic pH adjuster includes acetic acid.

14. The hydroxyapatite-collagen material according to claim 12, characterized in that The alkaline pH adjuster includes an aqueous solution of NaOH.

15. The hydroxyapatite-collagen material according to claim 14, characterized in that The mass percentage of the aqueous solution of NaOH is 1-20%.

16. A method for preparing the hydroxyapatite-collagen material according to any one of claims 1 to 15, characterized in that: The preparation method comprises the following steps: Through the amidation reaction between the carboxyl groups in alginate and the amino groups in collagen fibers, alginate is grafted onto the collagen fibers to obtain alginate-collagen fibers; Calcium ions and phosphate ions are reacted on the alginate-collagen fibers to form hydroxyapatite by an in-situ deposition method; The hydroxyapatite-collagen material is obtained by cross-linking the calcium ions in the hydroxyapatite with the alginate-collagen fibers; Wherein, the amidation reaction specifically comprises the following steps: (a) mixing alginic acid and a halogenating agent to cause a halogenation reaction to obtain a halogenated product; (b) The halogenated product is reacted with collagen to obtain alginate-collagen fibers.

17. The method for preparing the hydroxyapatite-collagen material according to claim 16, characterized in that: In step (a), the temperature of the halogenation reaction is 10-30° C., and the time of the halogenation reaction is 1-3 h.

18. The method for preparing the hydroxyapatite-collagen material according to claim 16, characterized in that: In step (a), the halogenation reaction needs to be carried out in a reaction system with a pH of 8-9.

19. The method for preparing the hydroxyapatite-collagen material according to claim 16, characterized in that: In step (a), the pH of the halogenation reaction is adjusted to 8-9 using NaOH solution.

20. The method for preparing the hydroxyapatite-collagen material according to claim 16, characterized in that: In step (b), the reaction temperature is 40-60° C., and the reaction time is 7-9 h.

21. The method for preparing the hydroxyapatite-collagen material according to claim 16, characterized in that: Before the calcium ions and phosphate ions react, the pH of the alginate-collagen fiber system needs to be adjusted to 6.8-7.

2.

22. The method for preparing the hydroxyapatite-collagen material according to claim 16, characterized in that: The in-situ deposition method specifically includes the following steps: simultaneously adding a calcium chloride solution and a sodium phosphate solution to the alginate-collagen fiber system.

23. The method for preparing the hydroxyapatite-collagen material according to claim 22, characterized in that: The concentration of the calcium chloride solution is 0.18-0.22 mol / L.

24. The method for preparing the hydroxyapatite-collagen material according to claim 22, characterized in that: The concentration of the sodium phosphate solution is 0.22-0.26 mol / L.

25. The method for preparing the hydroxyapatite-collagen material according to claim 22, characterized in that: The calcium chloride solution is added at a rate of 1.7-1.9 g / min.

26. The method for preparing the hydroxyapatite-collagen material according to claim 22, characterized in that: The sodium phosphate solution is added at a rate of 1.5-1.7 g / min.

27. The method for preparing the hydroxyapatite-collagen material according to claim 22, characterized in that: The difference in the dropping speed of the calcium chloride solution and the sodium phosphate solution is 0.1-0.3 g / min.

28. The method for preparing the hydroxyapatite-collagen material according to claim 16, characterized in that: The cross-linking reaction is carried out by stirring.

29. The method for preparing the hydroxyapatite-collagen material according to claim 16, characterized in that: The temperature of the cross-linking reaction is 80-120° C., and the time of the cross-linking reaction is 2-4 h.

30. The method for preparing the hydroxyapatite-collagen material according to claim 16, characterized in that: The preparation method comprises the following steps: (1) Phosphorus pentachloride is added dropwise to an aqueous solution containing alginic acid, and the reaction is carried out at 10-30°C for 1-3 hours, and a pH regulator is used during the reaction to maintain the pH of the reaction system between 8 and 9; (2) Add collagen slurry to the reaction solution obtained in step (1) and react at 40-60°C for 7-9 hours; (3) After adjusting the pH of the reaction solution obtained in step (2) to 6.8-7.2 using a pH regulator, calcium chloride solution is added dropwise at a rate of 1.7-1.9 g / min and sodium phosphate solution is added dropwise at a rate of 1.5-1.7 g / min, and the reaction is continued at 10-30°C for 2-4 hours, and dried to obtain the hydroxyapatite-collagen material.

31. Use of the hydroxyapatite-collagen material according to any one of claims 1 to 15 in preparing a bone filling material.

Images