Mineralized collagen hydrogel microspheres, preparation method thereof and bone repair material

Abstract

The invention relates to the technical field of medical instruments, and discloses mineralized collagen hydrogel microspheres, a preparation method thereof and a bone repair material, the mineralized collagen hydrogel microspheres are prepared from mineralized collagen and sodium alginate composite liquid through electrospray, sodium alginate serves as a carrier of mineralized collagen, the degradation rate of the mineralized collagen is remarkably reduced, and the bone repair effect is improved. Meanwhile, the gel microspheres also have a higher specific surface area, so that the attached cells can quickly form tissues; according to the bone repair material, normal saline and mineralized collagen hydrogel microspheres are used, mineralized collagen in the mineralized collagen hydrogel microspheres can remarkably improve the corrosion resistance of sodium alginate in the normal saline, and the slow release effect of hydrogel in the mineralized collagen hydrogel microspheres can be remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of medical devices, and in particular to mineralized collagen hydrogel microspheres, a preparation method thereof, and a bone repair material. Background Art

[0002] Soft tissue and cartilage are important load-bearing and motor-functional tissues in the human body. Cartilage is mainly composed of type II collagen and proteoglycans, and has excellent compressive and shock-absorbing properties, but its regeneration capacity is limited. Soft tissues such as tendons and ligaments are characterized by highly oriented type I collagen fiber bundles with anisotropic mechanical properties. In tissue engineering, biomimetic materials need to simulate the layered mineralization structure of cartilage and the fiber orientation characteristics of soft tissue, while regulating the release of calcium and phosphate ions to promote interface integration. The ideal repair material should have the lubricity of cartilage, the toughness of soft tissue, and the strength of bone tissue to match the functional requirements of the complex physiological environment.

[0003] As a biomimetic composite material, mineralized collagen has a structure similar to that of mineralized collagen in natural human tissues. It has excellent osteocompatibility and osteoinduction ability, and plays an important role in the repair of bone and cartilage tissue. Collagen provides a biomimetic microenvironment for cell adhesion and proliferation, while the mineral phase participates in regulating the key pathways of osteoblast differentiation by releasing calcium and phosphorus ions. In the field of cartilage repair, by balancing the ion concentration and mechanical stimulation in the microenvironment, stem cells are guided to differentiate in a directional manner and the production of chondrocytes is promoted. For the repair of soft tissues such as tendons and ligaments, mineralized collagen can match their fiber bundle structure, promoting cell infiltration and matrix remodeling while maintaining mechanical strength. However, traditional mineralized collagen materials are easily affected by tissue fluid and aggregated during application, resulting in local ion concentration imbalance. It is difficult to achieve precise matching of mineralization gradient and fiber orientation in dynamic mechanical environments such as tendons and ligaments. In addition, there is a lack of accurate simulation of the osteoblast-cartilage interface microenvironment, which limits its application potential in the regeneration of complex tissue interfaces.

[0004] Invention publication number CN117045861A discloses a titanium mineralized collagen hydrogel suitable for bone repair materials and its preparation method. Although the invention uses titanium mineralized collagen to improve the mechanical strength and antibacterial properties of the collagen hydrogel, its titanium mineralized collagen hydrogel cannot effectively simulate the calcium-phosphorus metabolism process of the natural bone matrix and is difficult to regulate the key signal pathways of osteogenic / chondrogenic differentiation; at the same time, its macroscopic block hydrogel structure has problems such as low drug loading efficiency and uncontrollable drug release, which limits the material's application in complex tissue repair. Summary of the Invention

[0005] In order to overcome the problems of low drug loading efficiency and uncontrollable drug release in the macro-block hydrogel structure in the prior art, which limit the application of the material in complex tissue repair, the present invention provides a mineralized collagen hydrogel microsphere and its preparation method, as well as a bone repair material. The mineralized collagen hydrogel microsphere is prepared by electrospraying a mineralized collagen sodium alginate composite liquid, and sodium alginate is used as a carrier of mineralized collagen. The addition of sodium alginate will form thicker collagen fibers and have Ca 2+ The mechanically enhanced hydrogel with capture ability significantly reduces the degradation rate of mineralized collagen and significantly improves the duration of action of mineralized collagen. At the same time, the gel microspheres also have a higher specific surface area, which is conducive to the rapid formation of tissues by cells after attachment.

[0006] The specific technical solutions of the present invention are: The invention discloses a mineralized collagen hydrogel microsphere. The raw materials include, by weight, 0.1 to 1.2 parts of mineralized collagen, 1 to 3 parts of sodium alginate and a buffer.

[0007] Preferably, the molecular weight of sodium alginate is 100,000 to 200,000; and the buffer is one of sodium chloride solution and Ringer's solution.

[0008] Preferably, the pH of the buffer is 7 to 7.4.

[0009] Preferably, the raw materials of mineralized collagen include collagen, a phosphorus source and a calcium source.

[0010] Preferably, the collagen is one or both of animal-derived collagen and recombinant collagen.

[0011] Preferably, the phosphorus source is one or more of phosphoric acid solution, disodium hydrogen phosphate, sodium dihydrogen phosphate and dipotassium hydrogen phosphate.

[0012] Preferably, the calcium source is one or more of calcium chloride, calcium hydroxide, calcium nitrate and calcium acetate.

[0013] The present invention adopts a biomimetic mineralization method to prepare mineralized collagen. Collagen will self-assemble in a solvent to form a fibrous structure. Calcium ions and phosphate ions continuously aggregate on the surface of collagen fibers and form calcium phosphate crystal nuclei. The calcium phosphate crystal nuclei continuously grow to form biomimetic mineralized collagen. Mineralized collagen is composed of orderly arranged collagen and nano-hydroxyapatite, which can highly simulate the structure of natural bone tissue and is safe and efficient. The collagen component in the present invention accelerates wound healing by promoting cell proliferation and differentiation, and the mineralized component synergistically promotes repair by regulating cell metabolism. The mineralized collagen complex formed by the two has a synergistic effect on cell proliferation and differentiation, significantly improving tissue regeneration efficiency.

[0014] A method for preparing the above-mentioned mineralized collagen hydrogel microspheres comprises the following steps: (1) dissolving collagen in a phosphorus source aqueous solution to prepare a first solution, dissolving dopamine hydrochloride in water to prepare a second solution, and adding the first solution and a calcium source aqueous solution to the second solution to react and prepare mineralized collagen; (2) Sodium alginate, mineralized collagen and a buffer are mixed to prepare a mineralized collagen sodium alginate complex, and the mineralized collagen sodium alginate complex is subjected to electrospray treatment to prepare mineralized collagen hydrogel microspheres.

[0015] Preferably, the pH of the second solution is adjusted to 7-11 using aqueous ammonia.

[0016] Preferably, the reaction conditions of step (1) include: temperature 37° C., stirring speed 500-900 rpm, titration flow rate 20-150 mL / min, and reaction time 2-24 h.

[0017] Preferably, the conditions for the electrospray treatment include: an extrusion rate of 0.5 to 2 mL / h, a distance between the needle and the cross-linking bath of 5 to 9 cm, an applied voltage of 7 to 13 kV, and an injection needle of 25 to 30 G.

[0018] Preferably, the cross-linking bath is a calcium chloride solution with a concentration of 1 to 3 wt%.

[0019] Preferably, the particle size of the mineralized collagen is 100 to 200 nm.

[0020] The present invention prepares mineralized collagen hydrogel microspheres, wherein the sodium alginate and mineralized collagen in the microspheres have excellent biocompatibility, low immune rejection reaction in the body, and are safe biological implants; the hydrogel microspheres of the present invention are spherical hydrogels, which are strong in toughness and soft in texture, have excellent lubricity and injectability when used, are less destructive to healthy tissues, are safe, and have a weak foreign body sensation; in addition, a preparation method of the above-mentioned mineralized collagen hydrogel microspheres is also provided, wherein the mineralized collagen and sodium alginate are dissolved in a buffer, and the mineralized collagen hydrogel microspheres are prepared by electrospray treatment; the above-mentioned operation process does not use toxic or harmful organic solvents, is safe and pollution-free; the electrospray treatment can control the particle size of the hydrogel microspheres and can ensure that the particle size of the microspheres is uniform; the method can be produced in large quantities, the production process is non-toxic and harmless, and the degree of industrialization is high.

[0021] A bone repair material, the raw materials of which include the above-mentioned mineralized collagen hydrogel microspheres and physiological saline.

[0022] Preferably, the bone repair material contains 10-20% of mineralized collagen hydrogel microspheres per milliliter of normal saline.

[0023] The present invention also provides a bone repair material. The liquid carrier of the bone repair material of the present invention adopts isotonic solvent physiological saline. The present invention has found that the anti-corrosion effect of hydrogel microspheres formed by the use of sodium alginate and mineralized collagen in combination with physiological saline is significantly increased, and the sustained release effect of sodium alginate can be significantly increased. When the mineralized collagen sodium alginate hydrogel microspheres are used to load other substances, the release time of the sodium alginate load can be significantly increased.

[0024] Compared with the existing technology, this application has the following technical effects: (1) The mineralized collagen hydrogel microspheres provided by the present invention have a longer repair time than mineralized collagen; (2) The collagen component of the present invention accelerates wound healing by promoting cell proliferation and differentiation, and the mineralized component synergistically promotes repair by regulating cell metabolism. The mineralized collagen complex formed by the two has a synergistic effect on cell proliferation and differentiation, significantly improving tissue regeneration efficiency; (3) The preparation method of the mineralized collagen hydrogel microspheres provided by the present invention is non-toxic, harmless, green and safe, can control the uniformity of the microsphere particle size, and can be mass-produced with a high degree of industrialization; (4) The mineralized collagen of the present invention can improve the anti-corrosion effect of sodium alginate on physiological saline and can significantly improve the sustained-release ability of mineralized collagen sodium alginate hydrogel microspheres in physiological saline. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is an SEM image of the freeze-dried mineralized collagen hydrogel microspheres prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0026] The present invention will be further described below with reference to the embodiments.

[0027] Example 1 A method for preparing mineralized collagen hydrogel microspheres comprises the following steps: (1) Dissolve 0.2 g of bovine type I collagen powder in a disodium hydrogen phosphate solution (3.41 g of disodium hydrogen phosphate in 800 mL of water) and stir in the dark to prepare a first solution. The stirring conditions are: stirring temperature 37°C, stirring speed 400 rpm, and stirring time 10 h. (2) Dissolve 4.44 g of calcium source (calcium chloride) in 1000 mL of water and stir until dissolved to prepare a calcium source aqueous solution. The stirring conditions are: stirring speed 400 rpm, stirring time 30 min; (3) adding 0.3152 g of dopamine hydrochloride buffer to 10 mL of water and adjusting the pH to 9-10 using aqueous ammonia to prepare a second solution; (4) The second solution was heated and stirred at a stirring speed of 600 rpm and a temperature of 37°C, and the first solution and the calcium source solution (the mass ratio of calcium atoms to phosphorus atoms was 1.8) were titrated into the second solution at a rate of 50 mL / min. After the titration, the solution was protected from light and kept heated and stirred for 20 to 24 hours. After the stirring, the solution was allowed to stand for 1 hour and the supernatant was removed to obtain a crude mineralized collagen precipitate. The crude mineralized collagen was then centrifuged and washed three times at a speed of 3000 rpm, using water for washing. After washing, the solution was cooled and then cooled. Freeze dryer at -50°C and grind to produce mineralized collagen powder, the particle size of which is 300-500 nm; (5) add 1 g of sodium alginate to 100 mL of buffer (0.9% sodium chloride in calcium-free saline) and heat and stir at 600 rpm and 40°C until dissolved, the viscosity of sodium alginate is 200-500 Pa.s, then add 0.4 g of mineralized collagen powder and stir at 800 rpm for 6 h to produce a mineralized collagen-sodium alginate complex; (6) Dissolve 1.665 g of calcium chloride in 100 mL of buffer (0.9% sodium chloride in calcium-free saline) to prepare a crosslinking solution; (7) The cross-linking solution was injected into the cross-linking bath of the electrospray machine, and then the mineralized collagen sodium alginate complex was injected into the syringe of the electrospray machine for electrospray treatment. The conditions of the electrospray treatment were as follows: injection speed 0.5 mL / h, distance between the needle and the cross-linking bath 5 cm, applied voltage 10 kV, injection needle 28G, microspheres were aged in the cross-linking bath for 1 h, the microspheres were rinsed 3 times with sodium chloride solution, and then the microspheres were placed in a freeze dryer and dried at -50 ° C for 48 h to prepare mineralized collagen hydrogel microspheres, and the mineralized collagen hydrogel microspheres were stored at 4 ° C.

[0028] A bone repair material comprises the above-mentioned mineralized collagen hydrogel microspheres and physiological saline solution, wherein each milliliter of the physiological saline solution contains 20% of the mineralized collagen hydrogel microspheres.

[0029] Example 2 A method for preparing mineralized collagen hydrogel microspheres comprises the following steps: (1) 0.2 g of bovine type I collagen powder was dissolved in sodium dihydrogen phosphate solution (2.88 g of sodium dihydrogen phosphate was dissolved in 800 mL of water) and stirred in the dark to prepare the first solution. The stirring conditions were: stirring temperature 37°C, stirring speed 400 rpm, and stirring time 10 h. (2) Dissolve 4.44 g of calcium source (calcium chloride) in 1000 mL of water and stir until dissolved to prepare a calcium source aqueous solution. The stirring conditions are: stirring speed 400 rpm, stirring time 30 min; (3) adding 0.3152 g of dopamine hydrochloride buffer to 10 mL of water and adjusting the pH to 10-11 using aqueous ammonia to prepare a second solution; (4) The second solution was heated and stirred at a stirring speed of 600 rpm and a temperature of 37°C, and the first solution and the calcium source solution (the mass ratio of calcium atoms to phosphorus atoms was 1.6) were co-titrated into the second solution at a rate of 100 mL / min. After the titration, the solution was protected from light and kept heated and stirred for 10-12 hours. After the stirring, the solution was allowed to stand for 1 hour and the supernatant was removed to obtain a crude mineralized collagen precipitate. The crude mineralized collagen was then centrifuged and washed three times at a speed of 3000 rpm using water. After washing, the crude mineralized collagen was dried at -50°C in a freeze dryer and then ground to obtain a mineralized collagen powder. The particle size of the mineralized collagen powder was 200-300 nm. (5) 1 g of sodium alginate was added to 100 mL of buffer (0.9% sodium chloride in calcium-free saline) and heated and stirred at 600 rpm and 40°C until dissolved. The viscosity of the sodium alginate was 200-500 Pa.s. 0.4 g of mineralized collagen powder was then added and stirred at 800 rpm for 6 h to prepare a mineralized collagen-sodium alginate complex. (6) Dissolve 1.665 g of calcium chloride in 100 mL of buffer (0.9% sodium chloride in calcium-free saline) to prepare a crosslinking solution; (7) The cross-linking solution was injected into the cross-linking bath of the electrospray machine, and then the mineralized collagen sodium alginate complex was injected into the syringe of the electrospray machine for electrospray treatment. The conditions of the electrospray treatment were: injection speed 0.5 mL / h, distance between the needle and the cross-linking bath 5 cm, applied voltage 10 kV, injection needle 28G, microspheres were aged in the cross-linking bath for 1 h, the microspheres were rinsed 3 times with sodium chloride solution, and then the microspheres were placed in a freeze dryer and dried at -50 ° C for 48 h to prepare mineralized collagen hydrogel microspheres, and the mineralized collagen hydrogel microspheres were stored at 4 ° C.

[0030] A bone repair material comprises the above-mentioned mineralized collagen hydrogel microspheres and physiological saline solution, wherein each milliliter of the physiological saline solution contains 15% of the mineralized collagen hydrogel microspheres.

[0031] Example 3 A method for preparing mineralized collagen hydrogel microspheres comprises the following steps: (1) 0.2 g of bovine type I collagen powder was dissolved in a disodium hydrogen phosphate solution (4.18 g of dipotassium hydrogen phosphate was dissolved in 800 mL of water) and stirred in the dark to prepare a first solution. The stirring conditions were: stirring temperature 37°C, stirring speed 400 rpm, and stirring time 10 h. (2) Dissolve 1.11 g of calcium source (calcium chloride) in 1000 mL of water and stir until dissolved to prepare a calcium source aqueous solution. The stirring conditions are: stirring speed 400 rpm, stirring time 30 min; (3) adding 0.3152 g of dopamine hydrochloride buffer to 10 mL of water and adjusting the pH to 9-10 using aqueous ammonia to prepare a second solution; (4) The second solution was heated and stirred at a stirring speed of 600 rpm and a temperature of 37°C, and the first solution and the calcium source solution (the mass ratio of calcium atoms to phosphorus atoms was 1.5) were titrated into the second solution at a rate of 30 mL / min. After the titration, the solution was protected from light and kept heated and stirred for 10 h. After the stirring, the solution was allowed to stand for 1 h and the supernatant was removed to obtain a crude mineralized collagen precipitate. The crude mineralized collagen was then centrifuged and washed three times at a speed of 3000 rpm. Water was used for washing, and then the solution was frozen. After drying in a dryer at -50°C, the mixture was ground into a mineralized collagen powder having a particle size of 100 to 200 nm. (5) 1 g of sodium alginate was added to 100 mL of a buffer (0.9% sodium chloride in calcium-free saline) and heated and stirred at 600 rpm and 40°C until dissolved. The viscosity of the sodium alginate was 200 to 500 Pa.s. 0.4 g of the mineralized collagen powder was then added and stirred at 800 rpm for 6 h to prepare a mineralized collagen-sodium alginate complex. (6) Dissolve 1.665 g of calcium chloride in 100 mL of buffer (0.9% sodium chloride in calcium-free saline) to prepare a crosslinking solution; (7) The cross-linking solution was injected into the cross-linking bath of the electrospray machine, and then the mineralized collagen sodium alginate complex was injected into the syringe of the electrospray machine for electrospray treatment. The conditions of the electrospray treatment were as follows: injection speed 0.5 mL / h, distance between the needle and the cross-linking bath 5 cm, applied voltage 10 kV, injection needle 28G, microspheres were aged in the cross-linking bath for 1 h, the microspheres were rinsed 3 times with sodium chloride solution, and then the microspheres were placed in a freeze dryer and dried at -50 ° C for 48 h to prepare mineralized collagen hydrogel microspheres, and the mineralized collagen hydrogel microspheres were stored at 4 ° C.

[0032] A bone repair material comprises the above-mentioned mineralized collagen hydrogel microspheres and physiological saline solution, wherein each milliliter of the physiological saline solution contains 10% of the mineralized collagen hydrogel microspheres.

[0033] Example 4 A method for preparing mineralized collagen hydrogel microspheres comprises the following steps: (1) Dissolve 0.2 g of bovine type I collagen powder in dipotassium hydrogen phosphate solution (2.88 g of sodium dihydrogen phosphate dissolved in 800 mL of water) and stir in the dark to prepare the first solution. The stirring conditions are: stirring temperature 37°C, stirring speed 400 rpm, and stirring time 4 h. (2) Dissolve 9.45 g of calcium source (calcium nitrate) in 1000 mL of water and stir until dissolved to prepare a calcium source aqueous solution. The stirring conditions are: stirring speed 400 rpm, stirring time 1 h; (3) 0.3152 g of dopamine hydrochloride buffer solution was added to 10 mL of water and the pH was adjusted to 7-8 using ammonia water to prepare a second solution; (4) the second solution was heated and stirred at a stirring speed of 600 rpm and a temperature of 37°C, and the first solution and a calcium source solution (the mass ratio of calcium atoms to phosphorus atoms was 1.8) were titrated into the second solution at a rate of 50 mL / min. After the titration, the solution was protected from light and heated and stirred for 2-4 h; after the stirring was completed, the solution was allowed to stand for 1 h and the supernatant was removed to obtain a crude mineralized collagen precipitate. The crude mineralized collagen was then stirred at 3000 rpm for 1 h. The mixture was centrifuged and washed three times with water. After washing, it was dried in a freeze dryer at -50°C and then ground to produce mineralized collagen powder. The particle size of the mineralized collagen powder was 25-50 nm. (5) 1 g of sodium alginate was added to 100 mL of buffer (0.9% sodium chloride in calcium-free saline) and heated and stirred at 600 rpm and 40°C until dissolved. The viscosity of sodium alginate was 200-500 Pa.s. Then 0.4 g of mineralized collagen powder was added and stirred at 800 rpm for 6 h to produce a mineralized collagen-sodium alginate complex. (6) Dissolve 1.665 g of calcium chloride in 100 mL of buffer (0.9% sodium chloride in calcium-free saline) to prepare a crosslinking solution; (7) The cross-linking solution was injected into the cross-linking bath of the electrospray machine, and then the mineralized collagen sodium alginate complex was injected into the syringe of the electrospray machine for electrospray treatment. The conditions of the electrospray treatment were as follows: injection speed 0.5 mL / h, distance between the needle and the cross-linking bath 5 cm, applied voltage 10 kV, injection needle 28G, microspheres were aged in the cross-linking bath for 1 h, the microspheres were rinsed 3 times with sodium chloride solution, and then the microspheres were placed in a freeze dryer and dried at -50 ° C for 48 h to prepare mineralized collagen hydrogel microspheres, and the mineralized collagen hydrogel microspheres were stored at 4 ° C.

[0034] A bone repair material comprises the above-mentioned mineralized collagen hydrogel microspheres and physiological saline solution, wherein each milliliter of the physiological saline solution contains 20% of the mineralized collagen hydrogel microspheres.

[0035] Example 5 A method for preparing mineralized collagen hydrogel microspheres comprises the following steps: (1) Dissolve 0.2 g of bovine type I collagen powder in sodium dihydrogen phosphate solution (3.27 g of potassium dihydrogen phosphate dissolved in 800 mL of water) and stir in the dark to prepare the first solution. The stirring conditions are: stirring temperature 37°C, stirring speed 400 rpm, and stirring time 10 h. (2) Dissolve 7.05 g of calcium source (calcium acetate) in 1000 mL of water and stir until dissolved to prepare a calcium source aqueous solution. The stirring conditions are: stirring speed 400 rpm, stirring time 30 min; (3) 0.3152 g of dopamine hydrochloride buffer solution was added to 10 mL of water and the pH was adjusted to 7-8 using ammonia water to prepare a second solution; (4) the second solution was heated and stirred at a stirring speed of 600 rpm and a temperature of 37°C, and the first solution and a calcium source solution (the mass ratio of calcium atoms to phosphorus atoms was 1.8) were titrated into the second solution at a rate of 50 mL / min. After the titration, the solution was protected from light and heated and stirred for 6-8 hours; after the stirring was completed, the solution was allowed to stand for 1 hour and the supernatant was removed to obtain a crude mineralized collagen precipitate, and the crude mineralized collagen was stirred at 3000 rpm. The mixture was centrifuged and washed three times with water. After washing, it was dried in a freeze dryer at -50°C and then ground to produce mineralized collagen powder. The particle size of the mineralized collagen powder was 15-25 nm. (5) 1 g of sodium alginate was added to 100 mL of buffer (0.9% sodium chloride in calcium-free saline) and heated and stirred at 600 rpm and 40°C until dissolved. The viscosity of sodium alginate was 200-500 Pa.s. Then 0.4 g of mineralized collagen powder was added and stirred at 800 rpm for 6 h to produce a mineralized collagen-sodium alginate complex. (6) Dissolve 1.665 g of calcium chloride in 100 mL of buffer (0.9% sodium chloride in calcium-free saline) to prepare a crosslinking solution; (7) The cross-linking solution was injected into the cross-linking bath of the electrospray machine, and then the mineralized collagen sodium alginate complex was injected into the syringe of the electrospray machine for electrospray treatment. The conditions of the electrospray treatment were as follows: injection speed 0.5 mL / h, distance between the needle and the cross-linking bath 5 cm, applied voltage 10 kV, injection needle 28G, microspheres were aged in the cross-linking bath for 1 h, the microspheres were rinsed 3 times with sodium chloride solution, and then the microspheres were placed in a freeze dryer and dried at -50 ° C for 48 h to prepare mineralized collagen hydrogel microspheres, and the mineralized collagen hydrogel microspheres were stored at 4 ° C.

[0036] A bone repair material comprises the above-mentioned mineralized collagen hydrogel microspheres and physiological saline solution, wherein each milliliter of the physiological saline solution contains 20% of the mineralized collagen hydrogel microspheres.

[0037] Example 6 A method for preparing mineralized collagen hydrogel microspheres comprises the following steps: (1) 0.2 g of bovine type I collagen powder was dissolved in a dipotassium hydrogen phosphate solution (4.18 g of dipotassium hydrogen phosphate was dissolved in 800 mL of water) and stirred in the dark to prepare a first solution. The stirring conditions were: stirring temperature 37°C, stirring speed 400 rpm, and stirring time 10 h. (2) Dissolve 2.96 g of calcium source (calcium hydroxide) in 1000 mL of water and stir until dissolved to prepare a calcium source aqueous solution. The stirring conditions are: stirring speed 400 rpm, stirring time 30 min; (3) 0.3152 g of dopamine hydrochloride buffer was added to 10 mL of water and the pH was adjusted to 6-7 using aqueous ammonia to prepare a second solution; (4) the second solution was heated and stirred at a stirring speed of 600 rpm and a temperature of 37°C, and the first solution and a calcium source solution (the mass ratio of calcium atoms to phosphorus atoms was 1.7) were titrated into the second solution at a rate of 50 mL / min. After the titration, the solution was protected from light and heated and stirred for 10-12 h; after the stirring, the solution was allowed to stand for 1 h and the supernatant was removed to obtain a crude mineralized collagen precipitate. The crude mineralized collagen was then centrifuged and washed three times at a speed of 3000 rpm using water. After washing, the crude mineralized collagen was dried at -50°C using a freeze dryer and then ground to obtain a mineralized collagen powder. The particle size of the mineralized collagen powder was 50-100 nm; (5) 1 g of sodium alginate was added to 100 mL of buffer (0.9% sodium chloride in calcium-free saline) and heated and stirred at 600 rpm and 40°C until dissolved. The viscosity of the sodium alginate was 200-500 Pa.s. 0.4 g of mineralized collagen powder was then added and stirred at 800 rpm for 6 h to prepare a mineralized collagen-sodium alginate complex. (6) Dissolve 1.665 g of calcium chloride in 100 mL of buffer (0.9% sodium chloride in calcium-free saline) to prepare a crosslinking solution; (7) The cross-linking solution was injected into the cross-linking bath of the electrospray machine, and then the mineralized collagen sodium alginate complex was injected into the syringe of the electrospray machine for electrospray treatment. The conditions of the electrospray treatment were as follows: injection speed 0.5 mL / h, distance between the needle and the cross-linking bath 5 cm, applied voltage 10 kV, injection needle 28G, microspheres were aged in the cross-linking bath for 1 h, the microspheres were rinsed 3 times with sodium chloride solution, and then the microspheres were placed in a freeze dryer and dried at -50 ° C for 48 h to prepare mineralized collagen hydrogel microspheres, and the mineralized collagen hydrogel microspheres were stored at 4 ° C.

[0038] A bone repair material comprises the above-mentioned mineralized collagen hydrogel microspheres and physiological saline solution, wherein each milliliter of physiological saline contains 20% of mineralized collagen hydrogel microspheres.

[0039] Example 7 A method for preparing mineralized collagen hydrogel microspheres comprises the following steps: (1) 0.2 g of bovine type I collagen powder was dissolved in a dipotassium hydrogen phosphate solution (4.18 g of dipotassium hydrogen phosphate was dissolved in 800 mL of water) and stirred in the dark to prepare a first solution. The stirring conditions were: stirring temperature 37°C, stirring speed 400 rpm, and stirring time 10 h. (2) Dissolve 9.45 g of calcium source (calcium nitrate) in 1000 mL of water and stir until dissolved to prepare a calcium source aqueous solution. The stirring conditions are: stirring speed 400 rpm, stirring time 30 min; (3) adding 0.3152 g of dopamine hydrochloride buffer to 10 mL of water and adjusting the pH to 10-11 using aqueous ammonia to prepare a second solution; (4) The second solution was heated and stirred at a stirring speed of 600 rpm and a temperature of 37°C, and the first solution and the calcium source solution (the mass ratio of calcium atoms to phosphorus atoms was 1.7) were co-titrated into the second solution at a rate of 100 mL / min. After the titration, the solution was protected from light and kept heated and stirred for 10-12 hours. After the stirring, the solution was allowed to stand for 1 hour and the supernatant was removed to obtain a crude mineralized collagen precipitate. The crude mineralized collagen was then centrifuged and washed three times at a speed of 3000 rpm using water. After washing, the crude mineralized collagen was dried at -50°C in a freeze dryer and then ground to obtain a mineralized collagen powder. The particle size of the mineralized collagen powder was 50-100 nm. (5) 1 g of sodium alginate was added to 100 mL of buffer (0.9% sodium chloride in calcium-free saline) and heated and stirred at 600 rpm and 40°C until dissolved. The viscosity of the sodium alginate was 200-500 Pa.s. 0.4 g of mineralized collagen powder was then added and stirred at 800 rpm for 6 h to prepare a mineralized collagen-sodium alginate complex. (6) Dissolve 1.665 g of calcium chloride in 100 mL of buffer (0.9% sodium chloride in calcium-free saline) to prepare a crosslinking solution; (7) The cross-linking solution was injected into the cross-linking bath of the electrospray machine, and then the mineralized collagen sodium alginate complex was injected into the syringe of the electrospray machine for electrospray treatment. The conditions of the electrospray treatment were as follows: injection speed 0.5 mL / h, distance between the needle and the cross-linking bath 5 cm, applied voltage 10 kV, injection needle 28G, microspheres were aged in the cross-linking bath for 1 h, the microspheres were rinsed 3 times with sodium chloride solution, and then the microspheres were placed in a freeze dryer and dried at -50 ° C for 48 h to prepare mineralized collagen hydrogel microspheres, and the mineralized collagen hydrogel microspheres were stored at 4 ° C.

[0040] A bone repair material comprises the above-mentioned mineralized collagen hydrogel microspheres and physiological saline solution, wherein each milliliter of the physiological saline solution contains 20% of the mineralized collagen hydrogel microspheres.

[0041] Example 8: A method for preparing mineralized collagen hydrogel microspheres comprises the following steps: (1) 0.2 g of bovine type I collagen powder was dissolved in 85% phosphoric acid solution (1.65 ml of 85% phosphoric acid in 800 ml of water) and stirred in the dark to prepare the first solution. The stirring conditions were: stirring temperature 37°C, stirring speed 400 rpm, and stirring time 10 h. (2) Dissolve 4.44 g of calcium source (calcium chloride) in 1000 mL of water and stir until dissolved to prepare a calcium source aqueous solution. The stirring conditions are: stirring speed 400 rpm, stirring time 30 min; (3) 0.3152 g of dopamine hydrochloride buffer was added to 10 mL of water and the pH was adjusted to 8-9 using aqueous ammonia to prepare a second solution; (4) the second solution was heated and stirred at a stirring speed of 600 rpm and a temperature of 37°C, and the first solution and a calcium source solution (the mass ratio of calcium atoms to phosphorus atoms was 1.6) were titrated into the second solution at a rate of 100 mL / min. After the titration, the solution was protected from light and heated and stirred for 10 h; after the stirring was completed, the solution was allowed to stand for 1 h and the supernatant was removed to obtain a crude mineralized collagen precipitate. The crude mineralized collagen was then centrifuged and washed three times at a speed of 3000 rpm using water. After washing, the crude mineralized collagen was dried at -50°C using a freeze dryer and then ground to obtain a mineralized collagen powder. The particle size of the mineralized collagen powder was 150-250 nm; (5) 1 g of sodium alginate was added to 100 mL of buffer (0.9% sodium chloride in calcium-free saline) and heated and stirred at 600 rpm and 40°C until dissolved. The viscosity of the sodium alginate was 200-500 Pa.s. 0.4 g of mineralized collagen powder was then added and stirred at 800 rpm for 6 h to prepare a mineralized collagen-sodium alginate complex. (6) Dissolve 1.665 g of calcium chloride in 100 mL of buffer (0.9% sodium chloride in calcium-free saline) to prepare a crosslinking solution; (7) The cross-linking solution was injected into the cross-linking bath of the electrospray machine, and then the mineralized collagen sodium alginate complex was injected into the syringe of the electrospray machine for electrospray treatment. The conditions of the electrospray treatment were as follows: injection speed 0.5 mL / h, distance between the needle and the cross-linking bath 5 cm, applied voltage 10 kV, injection needle 28G, microspheres were aged in the cross-linking bath for 1 h, the microspheres were rinsed 3 times with sodium chloride solution, and then the microspheres were placed in a freeze dryer and dried at -50 ° C for 48 h to prepare mineralized collagen hydrogel microspheres, and the mineralized collagen hydrogel microspheres were stored at 4 ° C.

[0042] A bone repair material comprises the above-mentioned mineralized collagen hydrogel microspheres and physiological saline solution, wherein each milliliter of the physiological saline solution contains 20% of the mineralized collagen hydrogel microspheres.

[0043] Comparative Example 1: Compared with Example 1, in Comparative Example 1, only sodium alginate was used to prepare the hydrogel microspheres, and the other conditions were the same as those in Example 1.

[0044] Comparative Example 2: Compared with Example 1, the particle size of the mineralized collagen in Comparative Example 2 is 1200-1500 nm, and the other conditions are the same as those in Example 1.

[0045] Comparative Example 3: Compared with Example 1, the particle size of the mineralized collagen in Comparative Example 3 is 5 to 10 nm, and the other conditions are the same as those in Example 1.

[0046] Test example The mineralized collagen hydrogel microspheres (20 mg) prepared in Examples 1 to 8 and Comparative Examples 1 to 3 were added to 80 ml of normal saline and shaken at 37° C. and 150 rpm. Samples were taken every 10 h to test the swelling rate and dissolution time of the sodium alginate hydrogel microspheres. The swelling rate was (mass of microspheres after swelling - mass of microspheres before swelling) / mass of microspheres before swelling, and the dissolution time was the time it took for the microspheres to completely rupture. The test results are shown in Table 1.

[0047] Table 1 Test results Mineralized collagen particle size (nm) Swelling rate (%) Dissolution time (h) Example 1 300～500 24 75 Example 2 200～300 20 85 Example 3 100～200 16 110 Example 4 25～50 13 140 Example 5 15～25 7 220 Example 6 50～100 12 160 Example 7 50～100 18 95 Example 8 150～250 20 85 Comparative Example 1 / 42 23 Comparative Example 2 1200～1500 37 68 Comparative Example 3 5～10 7 220 As shown in Table 1, in Comparative Example 1, no mineralized collagen was added to the sodium alginate. The results showed that the swelling rate of sodium alginate in normal saline reached 42%, and the dissolution time was 23 hours. In contrast, the swelling rate of the hydrogel microspheres composited with sodium alginate and mineralized collagen in normal saline in Example 1 was reduced to 24%, and the dissolution time was increased to 75 hours. The above results indicate that the composite of mineralized collagen and sodium alginate can significantly improve the anti-dissolution effect of sodium alginate in normal saline and prolong the sustained-release effect of sodium alginate.

[0048] It can also be seen from the results of Examples 1 to 8 that the particle size of mineralized collagen has a significant effect on the anti-corrosion effect of sodium alginate in normal saline. The smaller the particle size of the mineralized collagen, the smaller the swelling rate of sodium alginate in normal saline and the longer the dissolution time. Therefore, the dissolution time of sodium alginate can be controlled by controlling the particle size of the mineralized collagen during use, thereby preparing mineralized collagen sodium alginate hydrogels with different sustained-release time.

[0049] From the results of Comparative Examples 2 and 3, it can be seen that when the particle size of mineralized collagen exceeds 1500 nm, the anti-physiological saline dissolution effect of mineralized collagen on sodium alginate will not change significantly. When the particle size of mineralized collagen is less than nm, the anti-physiological saline dissolution effect of mineralized collagen on sodium alginate will also reach its limit and will not significantly change the anti-physiological saline dissolution effect of sodium alginate.

[0050] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A mineralized collagen hydrogel microsphere, characterized in that: The raw materials include, by mass, 0.1-1.2 parts of mineralized collagen, 1-3 parts of sodium alginate and a buffer.

2. The mineralized collagen hydrogel microspheres according to claim 1, characterized in that: The molecular weight of sodium alginate is 100,000 to 200,000; the buffer is one of sodium chloride solution and Ringer's solution.

3. The mineralized collagen hydrogel microspheres according to claim 1 or 2, characterized in that: The pH of the buffer is 7~7.

4.

4. The mineralized collagen hydrogel microspheres according to claim 3, characterized in that: The raw materials of mineralized collagen include collagen, a phosphorus source and a calcium source; the collagen is one or both of animal-derived collagen and recombinant collagen; the phosphorus source is one or more of phosphoric acid solution, disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate and potassium dihydrogen phosphate; the calcium source is one or more of calcium chloride, calcium hydroxide, calcium nitrate and calcium acetate.

5. A method for preparing mineralized collagen hydrogel microspheres according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) dissolving collagen in a phosphorus source aqueous solution to prepare a first solution, dissolving dopamine hydrochloride in water to prepare a second solution, and adding the first solution and a calcium source aqueous solution to the second solution to react and prepare mineralized collagen; (2) Sodium alginate, mineralized collagen and a buffer are mixed to prepare a mineralized collagen sodium alginate complex, and the mineralized collagen sodium alginate complex is subjected to electrospray treatment to prepare mineralized collagen hydrogel microspheres.

6. The preparation method according to claim 5, characterized in that: The pH of the second solution was adjusted to 6-12 using aqueous ammonia.

7. The preparation method according to claim 5, characterized in that: The reaction conditions of step (1) include: temperature 37°C, stirring speed 500-900 rpm, titration flow rate 20-150 mL / min, and reaction time 2-24 h.

8. The preparation method according to claim 5, characterized in that: The electrospray treatment conditions include: an extrusion rate of 0.01-2 mL / h, a distance between the needle and the cross-linking bath of 3-10 cm, an applied voltage of 4-20 kV, an injection needle of 21-30 G, and a particle size of the microspheres of 50-1000 μm.

9. The preparation method according to claim 8, characterized in that: The cross-linking bath is a calcium chloride solution with a concentration of 1-3 wt%.

10. A bone repair material, characterized in that: The raw materials include the mineralized collagen hydrogel microspheres according to any one of claims 1 to 4 and physiological saline.

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