Biomimetic mineralized II-type collagen hydrogel as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to biomimetic mineralized II-type collagen hydrogel as well as a preparation method and application thereof. The method comprises the following steps: gelling an acid solution of type II collagen in an alkaline environment to obtain type II collagen hydrogel; and dipping the type II collagen hydrogel in a mineralizing solution for mineralization to obtain the biomimetic mineralized type II collagen hydrogel. According to the method provided by the invention, the complex shape and nano-scale characteristics of the collagen fiber template can be accurately filled and copied, and the obtained biomimetic mineralized II-type collagen hydrogel can realize a tissue bionic effect. The biomimetic mineralized gel prepared by the invention has type I and type II amido bonds, the unmineralized collagenous fiber has a 67nm D-banding periodic structure, and hydroxyapatite crystals in the mineralized collagenous fiber are distributed along a long axis of collagen fibril composed of type II collagen, so that mineralization in the collagenous fiber is realized.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedical materials, and particularly relates to a biomimetic mineralized type II collagen hydrogel as well as a preparation method and application thereof. BACKGROUND

[0002] The tendon-bone interface is composed of four layers of continuously transitional tissues: bone-calcified fibrous cartilage-uncalcified fibrous cartilage-tendon. This gradient structure can alleviate stress concentration, but is difficult to regenerate spontaneously after injury, and is often replaced by scar tissue, resulting in a high rate of re-tear of 20% to 95%.

[0003] In recent years, the development of tissue engineering has brought new hope for tendon-bone regeneration and repair, but the current tissue engineering scaffold materials cannot achieve tissue biomimetic effect. SUMMARY

[0004] The application aims to provide a biomimetic mineralized type II collagen hydrogel as well as a preparation method and application thereof. The method provided by the application can accurately fill and copy the complex shape and nanoscale features of a collagen fiber template, and the obtained biomimetic mineralized type II collagen hydrogel can achieve tissue biomimetic effect.

[0005] In order to achieve the above-mentioned purpose, the application provides the following technical scheme: The application provides a preparation method of a biomimetic mineralized type II collagen hydrogel, comprising the following steps: gelling an acid solution of type II collagen in an alkaline environment to obtain a type II collagen hydrogel; immersing the type II collagen hydrogel in a mineralization solution to mineralize to obtain the biomimetic mineralized type II collagen hydrogel, wherein the mineralization solution comprises a calcium salt, a phosphate salt and a non-collagen protein mimic.

[0006] Preferably, the acid solution of type II collagen is an acetic acid solution of type II collagen; the concentration of type II collagen in the acid solution of type II collagen is greater than or equal to 1 mg / mL, and the pH value of acetic acid is 2 to 4.

[0007] Preferably, the alkaline environment is an ammonia gas environment, which is generated by volatilization of ammonia water, and the mass percentage of NH3 in the ammonia water is greater than or equal to 10%; the gelling time is greater than or equal to 1 h; and the gelling environment is a closed environment.

[0008] Preferably, the mineralization solution is formed by a calcium salt solution, a phosphate salt solution and a non-collagen protein mimic; the molar concentration of calcium ions in the calcium salt solution is 0.1 to 20 mmol / L, the molar concentration of phosphate ions in the phosphate salt solution is 0.1 to 30 mmol / L, and the volume ratio of the calcium salt solution to the phosphate salt solution is 1:1.

[0009] Preferably, the non-collagen protein mimetic comprises polyaspartic acid or polyacrylic acid, and the mass concentration of the non-collagen protein mimetic in the mineralization solution is 50-500 μg / mL.

[0010] Preferably, the volume ratio of the mineralization solution to the type II collagen hydrogel is ≥0.5, and the mineralization time is ≥1 h.

[0011] Preferably, the mass percentage of the inorganic substance in the biomimetic mineralized type II collagen hydrogel is 33-59%.

[0012] Preferably, after obtaining the biomimetic mineralized type II collagen hydrogel, the biomimetic mineralized type II collagen hydrogel is subjected to freeze-drying to obtain a sponge-state product of the biomimetic mineralized type II collagen hydrogel.

[0013] The application provides the biomimetic mineralized type II collagen hydrogel prepared by the preparation method.

[0014] The application provides application of the biomimetic mineralized type II collagen hydrogel in biomimetic functional materials.

[0015] The application provides a preparation method of a biomimetic mineralized type II collagen hydrogel, comprising the following steps: forming a type II collagen hydrogel by gelation of an acid solution of type II collagen in an alkaline environment; and mineralizing the type II collagen hydrogel in a mineralization solution to obtain the biomimetic mineralized type II collagen hydrogel, wherein the mineralization solution comprises a calcium salt, a phosphate salt and a non-collagen protein mimic. The application uses type II collagen as a raw material, first forms a type II collagen hydrogel in an alkaline environment, and then mineralizes the type II collagen hydrogel in a solution containing a non-collagen protein mimic, calcium ions and phosphate ions. The negatively charged soluble polymer molecules (non-collagen protein mimic) in the mineralization solution simulate the non-collagen protein in biological mineralization. The non-collagen protein mimic molecules combine with mineral-forming ions in the solution through electrostatic interaction, hydrogen bonding and other forces. Then the non-collagen protein mimic effectively stabilizes the structure-unstable amorphous calcium phosphate precursor by limiting the freedom of ions, and then enters the collagen fibril composed of type II collagen, combines with the negative charge group, and then dehydrates and phase changes into a hydroxyapatite crystal, and arranges along the long axis of the collagen fibril, thereby accurately filling and copying the complex shape and nanoscale characteristics of the collagen fibril template. In summary, the application uses the collagen fibril composed of type II collagen as a matrix, first forms a hydrogel in an alkaline environment, and then performs internal mineralization of the type II collagen fibril in a mineralization solution to uniformly deposit calcium phosphate minerals in the three-dimensional grid, thereby forming a composite gel product with a hierarchical porous structure, i.e., the biomimetic mineralized type II collagen hydrogel. The method provided by the application can accurately fill and copy the complex shape and nanoscale characteristics of the collagen fibril template, and the obtained biomimetic mineralized type II collagen hydrogel can achieve a tissue biomimetic effect. The data of the examples show that the biomimetic mineralized gel prepared by the application has type I and type II amide bonds (characteristics of type II collagen), the unmineralized collagen fibril has a 67nm D-banding periodic structure, the hydroxyapatite crystal is distributed along the long axis of the collagen fibril, and internal mineralization of the collagen fibril is achieved.

[0016] Further, in the application, the mass percentage content of inorganic matter in the biomimetic mineralized type II collagen hydrogel is 33-59%. The inorganic matter contained in the biomimetic mineralized type II collagen hydrogel prepared by the application is calcium phosphate mineral, and the calcium phosphate mineral is hydroxyapatite. By optimizing the mass percentage content of inorganic matter in the biomimetic mineralized type II collagen hydrogel, the application can achieve a more optimal tissue biomimetic effect. The biomimetic mineralized type II collagen hydrogel prepared by the application is partially mineralized or fully mineralized, and the mass content of inorganic matter in the biomimetic mineralized type II collagen hydrogel prepared by the application is 33%, which is close to the inorganic matter content of cartilage, and the mass content of inorganic matter is 59%, which is close to the inorganic matter content of bone.

[0017] Further, in the present application, the non-collagen protein mimetic includes polyaspartic acid and / or polyacrylic acid, and the mass concentration of the non-collagen protein mimetic in the mineralization solution is 50-500 μg / mL. In the present application, the non-collagen protein mimetic is used as a stabilizer in the mineralization solution, and by limiting the type and mass concentration of the stabilizer in the mineralization solution, efficient in-vivo mineralization of collagen II fibrils is achieved. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The appearance information of the unmineralized hydrogel and the biomimetic mineralized collagen hydrogel prepared in the examples, Figure 1 The upper left graph in the figure is in the unmineralized state, and the remaining graphs are in the states of mineralization for 2 days, 4 days and 6 days; Figure 2 The thermogravimetric analysis (n=3) graph of the biomimetic mineralized collagen film prepared in the examples; Figure 3 The transmission electron microscope characterization graph of the unmineralized collagen II hydrogel in the examples and the biomimetic mineralized collagen II hydrogel prepared in the examples; Figure 4 The infrared spectrum graph of the collagen II hydrogel. DETAILED DESCRIPTION

[0019] The present application provides a preparation method of a biomimetic mineralized collagen II hydrogel, comprising the following steps: Gelatinizing the acid solution of collagen II in an alkaline environment to obtain a collagen II hydrogel; Immersion of the collagen II hydrogel in a mineralization solution for mineralization to obtain the biomimetic mineralized collagen II hydrogel, wherein the mineralization solution comprises a calcium salt, a phosphate salt and a non-collagen protein mimetic.

[0020] In the present application, all the raw materials / components are commercially available products well known to those skilled in the art unless otherwise specified. The percentages in the present application are mass percentages unless otherwise specified. The solutions in the present application are aqueous solutions with water as the solvent unless otherwise specified, for example, the calcium chloride solution is a calcium chloride aqueous solution; the normal temperature and room temperature in the present application generally refer to a temperature of 15-25℃, and are generally defined as 25℃.

[0021] The application gels an acid solution of type II collagen in an alkaline environment to obtain a type II collagen hydrogel. In the embodiments of the application, the type II collagen can be a type II collagen sponge. In the application, the preparation method of the type II collagen sponge preferably comprises: first mixing cartilage and a first buffer solution to obtain first treated cartilage; second mixing the first treated cartilage and a second buffer solution to obtain a second mixed solution, centrifuging the second mixed solution to obtain a precipitate; mixing the precipitate and a pepsin solution for enzymolysis to obtain an enzymolysis solution; centrifuging the enzymolysis solution to obtain a supernatant; salt-analyzing the supernatant with a sodium chloride solution to obtain a salt-analyzed solution; centrifuging the salt-analyzed solution to obtain a type II collagen crude product precipitate; and freeze-drying the type II collagen crude product precipitate after dialysis to obtain the type II collagen sponge. In the application, the cartilage can be pig rib cartilage. Before the first mixing, the application preferably pretreats the cartilage, and the pretreatment preferably comprises: sequentially removing periosteum, washing, crushing and freeze-drying the cartilage. The first buffer solution is a sodium chloride and Tris-hydrochloric acid solution, and the molar concentration of sodium chloride in the sodium chloride and Tris-hydrochloric acid solution is preferably 3 mol / L. The molar concentration of Tris-hydrochloric acid in the sodium chloride and Tris-hydrochloric acid solution is preferably 0.05 mol / L. The dosage ratio of the cartilage to the first buffer solution is preferably 1 g:10 mL. The first mixing is performed at room temperature, and the first mixing is performed under stirring. The time of the first mixing is preferably 10-12 h. The second buffer solution is preferably a guanidine hydrochloride and Tris-hydrochloric acid solution, and the molar concentration of guanidine hydrochloride in the guanidine hydrochloride and Tris-hydrochloric acid solution is preferably 4 mol / L. The molar concentration of Tris-hydrochloric acid in the guanidine hydrochloride and Tris-hydrochloric acid solution is preferably 0.05 mol / L. The dosage ratio of the cartilage to the second buffer solution is preferably 1 g:10 mL. The second mixing is performed at room temperature, and the second mixing is performed under stirring. The time of the second mixing is preferably 10-12 h. The application controls the time of the second mixing to ensure that the insoluble proteins in the first treated cartilage are fully dissolved. The centrifugation temperature of the second mixed solution is preferably 2-4°C, the rotation speed is preferably 10,000-12,000 r / min, and the time is preferably 20-30 min. The pH value of the enzymolysis is preferably 3-4, and the pH value of the enzymolysis is preferably adjusted with an acetic acid solution, and the molar concentration of the acetic acid solution is preferably 0.5 mol / L. The time of the enzymolysis is preferably 16-18 h. The rotation speed of the centrifugation of the enzymolysis solution is preferably 10,000-12,000 r / min, and the time is preferably 20-30 min. The molar concentration of the sodium chloride solution used for the salt analysis is preferably 2-2.5 mol / L.The salting-out time is preferably 1-1.5 hours. The temperature for centrifuging the salting-out solution is preferably 2-4℃, and the rotation speed is preferably 10,000-12,000 r / min. The dialysis bag used in the dialysis is preferably a bag with a MD-12,000 dialysis bag surface. The dialysis time is preferably 24-48 hours. The freeze-drying is preferably freezing the collagen solution obtained by dialysis into ice blocks and then transferring the ice blocks to a freeze-drier for freeze-drying for 24-72 hours.

[0022] In the present application, the acid solution of type II collagen is preferably an acetic acid solution of type II collagen. The concentration of type II collagen in the acid solution of type II collagen is preferably ≥1 mg / mL, more preferably ≥3 mg / mL, and most preferably 10-20 mg / mL. The pH value of the acetic acid is preferably 2-4, and in the examples, the pH value can be 3.

[0023] In the present application, the alkaline environment is preferably an ammonia gas environment. The ammonia gas environment is generated by volatilization of ammonia water, and the mass percentage of NH3 in the ammonia water is preferably ≥10%.

[0024] In the present application, the gelation time (i.e., the time for forming a type II collagen hydrogel) is preferably ≥1 hour, and more preferably 3-4 hours. The gelation environment is preferably a closed environment.

[0025] In the present application, after the gelation, an initial gel is obtained, and the initial gel is preferably washed with water to obtain the type II collagen hydrogel. The water washing is preferably performed by soaking with deionized water. The water washing is preferably performed by soaking for multiple times, and each soaking time is ≥10 minutes. The number of water washing times is preferably ≥5.

[0026] After obtaining the type II collagen hydrogel, the type II collagen hydrogel is immersed in a mineralization solution to perform mineralization, thereby obtaining the biomimetic mineralized type II collagen hydrogel. The mineralization solution comprises a calcium salt, a phosphate salt, and a non-collagen protein mimic.

[0027] In the present application, the mineralization solution is preferably formed by a calcium salt solution, a phosphate salt solution, and a non-collagen protein mimic. The calcium salt solution is preferably a calcium chloride solution. The molar concentration of calcium ions in the calcium salt solution is preferably 0.1-20 mmol / L, more preferably 0.5-5 mmol / L, and further preferably 1-4 mmol / L.

[0028] In the present application, the phosphate solution is preferably a mixed solution of disodium hydrogen phosphate and sodium chloride. The molar concentration of phosphate ions in the phosphate solution is preferably 0.1-30 mmol / L, more preferably 5-30 mmol / L, further preferably 5-20 mmol / L, and most preferably 9-10 mmol / L, and in the examples, it can be 19 mmol / L. The concentration of sodium chloride in the phosphate solution is preferably 100-400 mmol / L, and in the examples, it can be 300 mmol / L.

[0029] In the present application, the volume ratio of the calcium salt solution to the phosphate solution is preferably 1:1.

[0030] In the present application, the non-collagen protein mimetic preferably includes polyaspartic acid or polyacrylic acid, and in the examples, it can be polyaspartic acid. The mass concentration of the non-collagen protein mimetic in the mineralization solution is preferably 50-500 µg / mL, more preferably 100-400 µg / mL, further preferably 200-300 µg / mL, and most preferably 240 µg / mL.

[0031] In the present application, the preparation method of the mineralization solution preferably includes mixing the calcium salt solution and the non-collagen protein mimetic solution, and then mixing with the phosphate solution. In the examples of the present application, the preparation method of the mineralization solution can be mixing the calcium chloride solution and the non-collagen protein mimetic solution, and then mixing with the mixed solution of disodium hydrogen phosphate and sodium chloride. In the present application, the preparation method of the mineralization solution using the above method can make the non-collagen protein mimetic stabilize the calcium ions as a stabilizer first, and then after mixing with the solution of disodium hydrogen phosphate and sodium chloride, the calcium ions can be slowly released to form amorphous calcium phosphate nanoclusters.

[0032] In the present application, the volume ratio of the mineralization solution to the type II collagen hydrogel is preferably ≥0.5, more preferably ≥1, further preferably ≥5, and most preferably 5-10. In the present application, when the volume ratio of the mineralization solution to the type II collagen hydrogel is within the above range, it can ensure that the amount of the mineralization solution is sufficient, and thus the inorganic content in the obtained product can meet the requirements of the biomimetic composition.

[0033] In the present application, the mineralization time is preferably ≥1 h, and more preferably ≥2 days. In the present application, the mineralization solution is preferably replaced once every 12 h during the mineralization process.

[0034] In the present application, after the mineralization is completed, the obtained mineralization product is preferably washed with deionized water ≥1 time, and more preferably ≥3 times, to obtain the biomimetic mineralized type II collagen hydrogel.

[0035] In the present application, after obtaining the biomimetic mineralized type II collagen hydrogel, the present application preferably further comprises freeze-drying the biomimetic mineralized type II collagen hydrogel to obtain a sponge state product of the biomimetic mineralized type II collagen hydrogel. The freeze-drying can be carried out in a freeze dryer. The freeze-drying time can be 3-5 days.

[0036] The present application provides the biomimetic mineralized type II collagen hydrogel prepared by the preparation method described in the above technical solution. The biomimetic mineralized type II collagen hydrogel prepared by the present application has type I and type II amide bonds (type II collagen characteristic), the un-mineralized collagen fibrils have a 67 nm D-banding periodic structure, and the hydroxyapatite crystals in the mineralized collagen fibrils are distributed along the long axis of the collagen fibrils, realizing the mineralization in the collagen fibers.

[0037] In the present application, the mass percentage content of inorganic matter in the biomimetic mineralized type II collagen hydrogel is preferably 33-59%.

[0038] The present application provides the application of the biomimetic mineralized type II collagen hydrogel described in the above technical solution in biomimetic functional materials.

[0039] The biomimetic mineralized type II collagen hydrogel provided by the present application can realize the regeneration and repair of the tendon-skeletal interface.

[0040] In order to further illustrate the present application, the technical solutions provided by the present application are described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0041] Example 1 Extraction of type II collagen: Take 100 g of pig rib cartilage, remove the periosteum and wash, then crush it with a blender after freeze-drying; Dissolve: Put the chopped cartilage into the prepared sodium chloride (3M) and Tris-hydrochloric acid solution (0.05M), add 10 mL: 1 g (solution: cartilage) ratio, stir at room temperature for 12 hours; Add the cartilage block treated with sodium chloride and Tris-hydrochloric acid solution into the prepared guanidine hydrochloride (4M) and Tris-hydrochloric acid (0.05M) solution, add 10 mL: 1 g ratio (solution: cartilage). Stir at room temperature for 12 hours to ensure that the insoluble proteins are fully dissolved. Then centrifuge at 12000 rpm for 20 minutes at 4°C, and take the precipitate; Add the precipitate into the pepsin solution, keep the pH of the enzymatic solution between 3.0-4.0 (0.5M acetic acid adjustment), and enzymatic for 16 hours; Transfer the enzymatic solution to a centrifuge tube and centrifuge at 12000 rpm for 30 minutes using a centrifuge to obtain the supernatant, which is the crude collagen protein solution; Gradient salting with sodium chloride (2.5M) solution for 1 hour, then centrifuge at 12000 rpm at 4°C to obtain collagen protein precipitate; Dialysis for 24-48 hours using a bag face MD-12000 dialysis bag; Freeze-drying: After dialysis, the collagen solution is frozen into ice blocks and transferred to a freeze-dryer for freeze-drying for 24-72 hours until dry type II collagen sponge is obtained.

[0042] Example 2 Preparation of type II collagen hydrogel: Dissolve the type II collagen sponge in acetic acid solution (pH=3) to prepare a 20mg / mL type II collagen solution, take 100µL and add it to the mold, diffuse in a closed ammonia environment at room temperature for 4h to form a gel, then take it out and wash it with deionized water for 5 times, 10min / time, to make its pH value 7, to obtain type II collagen hydrogel, the mass concentration of type II collagen hydrogel is 20mg / mL.

[0043] Preparation of mineralization solution: Mix 20mL calcium chloride solution (concentration of 3.34mM), 960µL 10mg / mL polyaspartic acid solution, and 20mL sodium hydrogen phosphate and sodium chloride mixed solution, and mix well, ready for use. The concentration of sodium hydrogen phosphate in the sodium hydrogen phosphate and sodium chloride mixed solution is 19mM, and the concentration of sodium chloride is 300mM. The volume ratio of calcium chloride solution to sodium hydrogen phosphate and sodium chloride mixed solution is 1:1, and the mass concentration of polyaspartic acid in the mineralization solution is 240µg / mL.

[0044] Mineralization of collagen hydrogel: the type II collagen hydrogel was immersed in 10 mL of mineralization solution (volume ratio of type II collagen hydrogel to mineralization solution was 1:5), and was transferred into a 37°C constant temperature incubator, and the mineralization solution was replaced every 12 hours, and the mineralization lasted for 2-6 days (specifically 2 days, 4 days, and 6 days), and after the mineralization was completed, the type II collagen hydrogel was washed with deionized water for 3 times, 10 min each time, to obtain the biomimetic mineralized type II collagen hydrogel.

[0045] Test Example 1 Figure 1 The appearance information of the non-mineralized type II collagen sponge prepared in Example 1 (the upper left graph in FIG. 1) and the biomimetic mineralized type II collagen hydrogel (the lower left graph, the upper right graph, and the lower right graph in FIG. 1) mineralized for 2 days, 4 days, and 6 days in Example 2 was characterized. Figure 1 Figure 1 The appearance information of the non-mineralized type II collagen sponge prepared in Example 1 (the upper left graph in FIG. 1) and the biomimetic mineralized type II collagen hydrogel (the lower left graph, the upper right graph, and the lower right graph in FIG. 1) mineralized for 2 days, 4 days, and 6 days in Example 2 was characterized.

[0046] Test Example 2 The biomimetic mineralized type II collagen hydrogel prepared in Example 2 was transferred to a freeze dryer, and after drying for 3 days, the biomimetic mineralized type II collagen sponge was obtained.

[0047] The biomimetic mineralized type II collagen sponge was subjected to thermogravimetric analysis, including the following steps: Step S1: the sample was placed in a 37°C oven for 3 days, and subjected to thermogravimetric analysis; Step S2: combustion conditions: 30-800°C, air environment; Step S3: the mass at 660°C was analyzed as the total mass of inorganic matter; Figure 2 The thermogravimetric analysis of the biomimetic mineralized type II collagen hydrogel prepared in Example 2, with inorganic matter content of 33 wt% (n=3), 53 wt% (n=3), and 59 wt% (n=3), was characterized.

[0048] Test Example 3 The biomimetic mineralized type II collagen hydrogel prepared in Example 2 was transferred to a freeze dryer, and after drying for 3 days, the biomimetic mineralized type II collagen sponge was obtained.

[0049] Step S1: 0.01 g of the type II collagen hydrogel prepared in Example 2 at a concentration of 20 mg / mL was weighed into 1 mL of ammonium bicarbonate solution (pH=7.8), and was grinded in a homogenizer, 16 s each time, repeated 4 times; Step S2: 2-fold and 4-fold dilutions were performed, 2 μL was dropped on a nickel mesh, and was tightly sealed with a parafilm, and was placed at room temperature for 3 days; Step S3: washed with water for 1 min, 50% alcohol for 1 min, and 100% alcohol for 1 min, and then was dried on filter paper; ​Step S4: Transmission electron microscope imaging, non-mineralized gel needs to be dyed: 5 μL drop of uranyl acetate, 10 s, dry before baking the lamp.

[0050] The above experimental steps are repeated using biomimetic mineralized type II collagen sponge as raw material.

[0051] Figure 3 The microstructure of non-mineralized and biomimetic mineralized collagen hydrogel is characterized by transmission electron microscopy. As shown in the upper part of Figure 3 , the self-assembled type II collagen fibrils have 67 nm periodic cross-striations (D-banding) with light and dark alternation, similar to natural collagen fibrils; in addition, as shown in the lower part of Figure 3 , the D-banding structure of type II collagen fibrils mineralized for 2 days, 4 days and 6 days disappears partially, hydroxyapatite crystals are formed in the type II collagen fibrils, forming an organic-inorganic composite structure, and the degree of mineralization is strengthened with the mineralization time.

[0052] Test Example 4 The type II collagen sponge prepared in Example 1 (non-mineralized) is dried at 37℃ for 3 days, and then analyzed by Fourier infrared spectroscopy AVATAR 370. The results are shown in Figure 4 , it can be seen that the type II collagen hydrogel has a stretching vibration peak of amide I band (3336 cm -1 ), and the absorption peak is the strongest, which is a sensitive region for the change of protein secondary structure. The characteristic absorption frequency of amide II band (1544 cm -1 ) is the absorption band produced by the superposition of α-helix, β-fold, turn and random coil. The 1200-1360 cm -1 band is attributed to amide III band, 1200-1400 cm -1 , which is the stretching vibration peak of N-H and the symmetric contraction vibration peak of COO-, which is an infrared spectroscopy feature that other proteins do not have. It may be due to the high content of glycine, hydroxyproline and proline in type II collagen, and the formation of a unique (Gly-Pro-Hyp) n sequence structure.

[0053] From the above examples, it can be seen that the biomimetic mineralized type II collagen hydrogel provided by the present application has an inorganic matter mass content of 33-59%, and the biomimetic mineralized type II collagen hydrogel prepared by the present application is partially mineralized or fully mineralized. The main feature of the structure of the biomimetic mineralized type II collagen hydrogel is the partially mineralized collagen fibril, and the inorganic matter distributed in the collagen fibril is the partially mineralized collagen fibril.

[0054] Although the above embodiments have been described in detail, it should be understood that these are only some embodiments of the present application, but not all embodiments, and other embodiments can be obtained without creativity on the basis of the above embodiments, and these embodiments all belong to the protection scope of the present application.

Claims

1. A method for preparing a biomimetic mineralized type II collagen hydrogel, characterized in that, Includes the following steps: Acidic solutions of type II collagen are gelled in an alkaline environment to obtain type II collagen hydrogels. The type II collagen hydrogel is immersed in a mineralizing solution for mineralization to obtain the biomimetic mineralized type II collagen hydrogel. The mineralizing solution includes calcium salts, phosphates, and non-collagen mimics.

2. The preparation method according to claim 1, characterized in that, The acidic solution of type II collagen is an acetic acid solution of type II collagen; the concentration of type II collagen in the acidic solution of type II collagen is ≥1 mg / mL, and the pH value of acetic acid is 2~4.

3. The preparation method according to claim 1 or 2, characterized in that, The alkaline environment is an ammonia environment, which is generated by the volatilization of ammonia water, and the mass percentage of NH3 in the ammonia water is ≥10%; the gelation time is ≥1 hour; the gelation environment is a closed environment.

4. The preparation method according to claim 1, characterized in that, The mineralization solution is formed from a calcium salt solution, a phosphate solution, and a non-collagen mimic; the molar concentration of calcium ions in the calcium salt solution is 0.1~20 mmol / L, the molar concentration of phosphate ions in the phosphate solution is 0.1~30 mmol / L, and the volume ratio of the calcium salt solution to the phosphate solution is 1:

1.

5. The preparation method according to claim 1 or 4, characterized in that, The non-collagen mimicry includes polyaspartic acid or polyacrylic acid, and the mass concentration of the non-collagen mimicry in the mineralization solution is 50~500 μg / mL.

6. The preparation method according to claim 1, characterized in that, The volume ratio of the mineralizing solution to the type II collagen hydrogel is ≥0.5; the mineralization time is ≥1h.

7. The preparation method according to claim 1 or 6, characterized in that, The inorganic content in the biomimetic mineralized type II collagen hydrogel is 33-59% by mass.

8. The preparation method according to claim 1, characterized in that, After obtaining the biomimetic mineralized type II collagen hydrogel, the process further includes freeze-drying the biomimetic mineralized type II collagen hydrogel to obtain a sponge-like product of the biomimetic mineralized type II collagen hydrogel.

9. A biomimetic mineralized type II collagen hydrogel prepared by any one of claims 1 to 8.

10. The application of the biomimetic mineralized type II collagen hydrogel according to claim 9 in biomimetic functional materials.

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