A photocrosslinkable biomimetic self-mineralizing hydrogel and its preparation method and application

Abstract

The present invention discloses a photo-crosslinked biomimetic self-mineralizing hydrogel, its preparation method and application, belonging to the technical field of medical preparations. The biomimetic self-mineralizing hydrogel of the present invention adopts a photo-crosslinking process and is prepared by subjecting a gel precursor solution obtained by compounding methacrylamide-modified type I collagen, methacrylate-modified carboxymethyl cellulose and a photoinitiator to thermosensitive self-assembly and photo-crosslinking. The preparation process of the process is simple and easy to implement, which is conducive to large-scale production. The biomimetic self-mineralizing hydrogel can simulate the microenvironment of the extracellular matrix during the biological mineralization of bone tissue, can self-recruit calcium and phosphorus for self-mineralization, has good osteogenic performance, provides a new strategy for the biomimetic repair of bone tissue defects, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of medical configurations, and particularly to a photo-crosslinked biomimetic self-mineralizing hydrogel, its preparation method and application. Background Art

[0002] Hydrogel materials have great application prospects in the field of biomedical engineering. Especially in the diagnosis and treatment of oral-related diseases, there is often a need for the repair of irregular bone defects. For example, diseases or diagnostic and treatment operations such as periodontitis, peri-implantitis, tooth extraction, etc. can cause irregular alveolar bone defects. By filling with hydrogel, it can play a role in hemostasis, anti-inflammation, and promoting bone repair.

[0003] The 3D microenvironment in which cells are located has an important regulatory effect on the fate of stem cells. As the main component of the extracellular matrix, collagen is widely used in the in vitro construction of tissue repair scaffolds. For example, the Chinese patent with the publication number CN114732958A selects type I collagen, an important structural protein, and carboxymethyl cellulose, a natural polysaccharide derivative, as raw materials, and can obtain a novel collagen-carboxymethyl cellulose / apatite composite material. The raw materials are rich in sources and all have good biocompatibility. No cross-linking agents (such as glutaraldehyde, carbodiimide salts, etc.) or adhesives are used during preparation, which can avoid the problem of potential toxic residues caused by additives. However, the material obtained by this invention lacks the ordered assembly of nanoapatite and collagen, cannot form a biomimetic collagen fiber inner-mineralized nanostructure, and requires freeze-drying and grinding through a sieve during preparation, and cannot achieve in-situ forming, which limits its application potential in clinical practice.

[0004] Amorphous calcium phosphate has broad applications in the biomedical field due to its good bioactivity, cell adhesion, controllable biodegradation rate, and excellent osteoconductivity (Amorphous calcium phosphate and its applications in biomedicine [J]. Journal of Inorganic Materials, 2007, 22(5): 775 - 782.). The Chinese patent with the publication number CN115957237A introduces the mineralized component of hybrid amorphous calcium phosphate into the tooth mineralization gel, which can provide a stable calcium and phosphorus source, induce tooth mineralization, and can incorporate metal ion salts and fluoride additives to adjust the hybridization degree of the amorphous calcium phosphate mineralized component, thereby endowing it with dual functions of mineralization and antibacterial, and can be used for the prevention and treatment of oral diseases such as dental white spots, early caries, and dentin hypersensitivity.

[0005] As a mineralized hard tissue, bone tissue has a precise hierarchical microstructure. At the nanoscale, collagen fibers and nano-hydroxyapatite crystals are orderly assembled to form mineralized collagen fibers. Amorphous calcium phosphate liquid precursor (ACP) is an important component of mineralization. In vitro, polyion, a non-collagen analogue, can be used to induce the formation of amorphous calcium phosphate liquid precursor (PILP), which can then mediate the in-fiber biomimetic mineralization of collagen fibers. However, PILP is usually synthesized at relatively low calcium and phosphorus concentrations, which is insufficient to provide the minerals required for bone healing at the macroscopic scale. Therefore, the collagen scaffold needs to be pre-mineralized, which has the disadvantages of long time consumption, cumbersome steps, low efficiency, and inability to form in situ, restricting its application potential in bone tissue engineering.

[0006] The mineralization form of fibers also has a significant impact on their application as biological scaffolds. Compared with the currently used ordinary external mineralization process or unmineralized collagen, the in-fiber mineralized collagen can endow bone tissue with tough and dynamic properties, enabling it to better perform various physiological functions. Using polyions such as polyacrylic acid to induce PILP can achieve the in-fiber mineralization of collagen fiber scaffolds and construct collagen scaffolds with in-fiber mineralization characteristics. However, the biocompatibility of polyions such as polyacrylic acid is limited, and they do not have the physicochemical properties of natural extracellular matrix and are not suitable as gel matrices for 3D cell culture, restricting their application in medical hydrogel materials.

[0007] Therefore, developing a green, safe, and widely sourced polyion stabilizer to jointly construct an in-situ self-mineralizing collagen-based gel with collagen that can spontaneously recruit mineral ions after implantation in the body is an ideal solution. Summary of the Invention

[0008] In view of the above-mentioned defects of the prior art, in the first aspect of the present invention, a preparation method of a biomimetic self-mineralizing hydrogel with simple operation is provided. Using a photocrosslinking process, it includes the following steps:

[0009] (1) Add methacrylic anhydride to an aqueous solution of carboxymethyl cellulose and mix to obtain a pre-reaction solution;

[0010] (2) Carry out an esterification reaction on the pre-reaction solution, and purify and dry the obtained crude product to obtain methacrylated carboxymethyl cellulose;

[0011] (3) Dissolve the methacrylated carboxymethyl cellulose in a buffer solution, further mix the obtained solution with a methacrylamidated type I collagen solution and a photoinitiator, and adjust the pH to be alkaline to obtain a gel precursor solution;

[0012] (4) Subject the gel precursor solution to thermosensitive self-assembly and photocrosslinking to obtain a biomimetic self-mineralizing hydrogel.

[0013] The biomimetic self-mineralizing hydrogel prepared by this method is made from a gel precursor solution obtained by compounding methacrylamide-modified type I collagen, methacrylate-esterified carboxymethyl cellulose, and a photoinitiator through thermosensitive self-assembly and photocrosslinking. Methacrylamide-modified type I collagen and methacrylate-esterified carboxymethyl cellulose achieve photocrosslinking by exciting the polymerization of methacryloyl groups under blue light irradiation.

[0014] Preferably, in the step (1), the molar ratio of the monomer of carboxymethyl cellulose to methacrylic anhydride is 1:1 to 100.

[0015] In the step, the sources of carboxymethyl cellulose are diverse and extensive. For example, carboxymethyl cellulose derived from plant cellulose, marine biological cellulose, bacterial cellulose, etc. can all be appropriately selected as raw materials to meet the requirements of the present invention.

[0016] Preferably, in the step (2), the esterification reaction is carried out in an environment with pH = 8 - 9, the temperature of the esterification reaction is 0 - 4 °C, and the reaction time is 4 - 24 h.

[0017] In the process, conventional methods in the art can be used to purify and dry the crude product. For example, according to the solubility of the product, the crude product solution can be placed in excess ethanol for alcohol precipitation, and the precipitate is collected and dialyzed in deionized water to thoroughly remove the reaction residues. In addition, freeze-drying under dark conditions is a drying method particularly suitable for this method.

[0018] Preferably, in the step (3), the pH of the buffer solution is the same as that of human blood, and its type includes one of TBS buffer solution, MES buffer solution, and HEPES buffer solution.

[0019] More preferably, when the buffer solution contains calcium ions and phosphate ions, the concentration of calcium ions is 4.5 mM - 9 M, and the molar ratio of calcium element to phosphorus element is 15:7 - 9.

[0020] Pre-mineralization requires first preparing a collagen scaffold, then soaking the collagen scaffold in a calcium-phosphorus solution for at least 2 - 3 days, and then culturing cells or implanting it into the body after mineralization is completed. However, the biomimetic self-mineralizing hydrogel of the present invention has the ability to spontaneously achieve internal mineralization, and it can be selected whether the buffer solution contains calcium and phosphorus elements according to its usage environment, providing more selectivity and flexibility for its application.

[0021] Preferably, in the step (3), the pH of the gel precursor solution is 8 - 9.

[0022] Preferably, in the step (3), by mass percentage, in the gel precursor solution, the content of methacrylamide-modified type I collagen is 0.1% to 10%, the content of methacrylate esterified carboxymethyl cellulose is 0.25% to 5%, and the content of the photoinitiator is 0.15% to 1%.

[0023] In the art, the conventional preparation method of the methacrylamide-modified type I collagen solution is to dissolve methacrylamide-modified type I collagen in an acetic acid solution. Additionally, based on the photoinitiated polymerization reaction of methacryloyl groups, photocrosslinking can use a suitable type of photoinitiator applicable to the art, and the selection is diverse. Commercially available products such as Irgacure 2959 photoinitiator can be used, or lithium phenyl(2,4,6-trimethylformyl)phosphate (LAP), riboflavin, etc. can also be used as photoinitiators.

[0024] Preferably, in the step (4), the method of thermosensitive self-assembly is as follows: The gel precursor solution is left standing in an environment of 35 - 38°C and a relative humidity of 90% - 100% until self-assembly is completed.

[0025] In the second aspect of the present invention, a biomimetic self-mineralizing hydrogel with good osteogenic properties is provided, which is prepared by the method of the first aspect of the present invention.

[0026] In the third aspect of the present invention, the application of the biomimetic self-mineralizing hydrogel of the second aspect of the present invention is provided, specifically as a raw material in the preparation of biological repair materials. For example, its application in the preparation of bone repair materials, periodontal tissue regeneration and repair materials, peri-implant tissue regeneration and repair materials, and extraction socket hemostasis and repair materials.

[0027] Based on the above technical solutions, the inventive concept and principle of the present invention lie in selecting carboxymethyl cellulose as the raw material. Carboxymethyl cellulose has characteristics similar to extracellular matrix polysaccharides, and at the same time contains polar groups such as carboxyl groups and hydroxyl groups, having good modifiability. In addition, carboxymethyl cellulose has good biological safety and has been approved by the US Food and Drug Administration (FDA). As a natural polysaccharide derivative with a wide range of sources, carboxymethyl cellulose is a polyanionic polyelectrolyte containing a large number of carboxyl groups, making it a highly potential amorphous calcium phosphate stabilizer. During the mineralization process of natural bone tissue, non-collagen proteins (NCPs) in the extracellular matrix can capture calcium ions from body fluids due to the presence of a large number of negatively charged acidic amino acid residues (such as polyaspartic acid and phosphorylated serine residues), mediating the formation of amorphous calcium phosphate (ACP). At the same time, the collagen-binding sites of non-collagen proteins can bind to collagen and guide amorphous calcium phosphate into the internal spaces of collagen fibers to achieve mineralization within the fibers. In vitro, polyanionic polyelectrolytes containing a large number of carboxyl groups, such as polyacrylic acid, are commonly used to simulate non-collagen proteins, capture calcium ions from supersaturated calcium-phosphate solutions to form polymer-induced liquid-phase precursors of amorphous calcium phosphate (PILP), and then mediate mineralization within collagen fibers.

[0028] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0029] The present invention provides a preparation method of a biomimetic self-mineralizing hydrogel, which has the characteristics of thermosensitive self-assembly and rapid photocrosslinking molding under human body temperature conditions, meeting the requirements of oral clinical diagnosis and treatment. The preparation process of the process is simple and easy to implement, which is conducive to large-scale production.

[0030] The present invention provides a biomimetic self-mineralizing hydrogel, which can simulate the microenvironment of the extracellular matrix during the biomineralization process of bone tissue, can self-recruit calcium and phosphorus for self-mineralization, and has good osteogenic performance.

[0031] The present invention also provides an application of the biomimetic self-mineralizing hydrogel, providing a new strategy for the biomimetic repair of bone tissue defects and having good application prospects. Description of the Drawings

[0032] Figure 1 1H nuclear magnetic resonance (NMR) spectra of methacrylated carboxymethyl cellulose (SCMA) and carboxymethyl cellulose (SCMC) for Example 1;

[0033] Figure 2 Infrared (IR) spectra of methacrylated carboxymethyl cellulose (SCMA) and carboxymethyl cellulose (SCMC) for Example 1;

[0034] Figure 3 Physical display diagrams of some steps in the preparation process of the biomimetic self-mineralizing hydrogel for Example 1;

[0035] Figure 4 Rheological detection image of the biomimetic self-mineralizing hydrogel of Example 1;

[0036] Figure 5 Transmission electron microscopy (TEM) image of the biomimetic self-mineralizing hydrogel of Example 1 soaked in simulated body fluid to achieve mineralization within collagen fibers;

[0037] Figure 6 Cryo-transmission electron microscopy (Cryo-TEM) image of methacrylated carboxymethyl cellulose of Example 2 recruiting calcium and phosphorus to form the PILP liquid precursor;

[0038] Figure 7 High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) image of methacrylated carboxymethyl cellulose prepared in Example 2 recruiting calcium and phosphorus to form the PILP liquid precursor, and the corresponding selected area diffraction (SAD) and energy-dispersive X-ray spectrometer (EDS) energy spectrum surface scan images;

[0039] Figure 8 Scanning electron microscopy (SEM) image of the in-situ self-mineralization morphology of the biomimetic self-mineralizing hydrogel of Example 2 after standing for 3 days;

[0040] Figure 9 CCK-8 detection results of the co-culture of the biomimetic self-mineralizing hydrogels (CS, CS-ACP) prepared in Example 1 and Example 2 with rat bone marrow mesenchymal stem cells (BMSCs) for 5 days;

[0041] Figure 10 Cytoskeleton staining images of the direct contact co-culture of the biomimetic self-mineralizing hydrogels (CS, CS-ACP) prepared in Example 1 and Example 2 with rat bone marrow mesenchymal stem cells (BMSCs) for 1 day;

[0042] Figure 11 PCR detection of the expression level of the osteogenic-related gene alkaline phosphatase (ALP) after 7 days of osteogenic induction in the direct contact co-culture of the biomimetic self-mineralizing hydrogels (CS, CS-ACP) prepared in Example 1 and Example 2 with rat bone marrow mesenchymal stem cells (BMSCs). Detailed implementation manners

[0043] The present invention will be further illustrated by the following examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0044] In the following examples:

[0045] Carboxymethyl cellulose, M.W. 90 kDa;

[0046] Dialysis bag, 14 kDa.

[0047] Example 1

[0048] A method for preparing a biomimetic self-mineralized hydrogel, the steps are as follows:

[0049] (1) Take 2 g of carboxymethyl cellulose (SCMC) and dissolve it in deionized water, stir overnight at room temperature to obtain an aqueous solution of 2 wt.% carboxymethyl cellulose; under an ice-water bath, continuously stir the solution at a rate of 800 rpm, take 34 g of methyl methacrylic anhydride, and dropwise add it to the aqueous solution of carboxymethyl cellulose under light-shielding conditions to obtain a pre-reaction solution;

[0050] (2) Place the pre-reaction solution at 4 °C, use a 5 M aqueous sodium hydroxide solution to adjust the pH of the pre-reaction solution to 8-9, continuously stir at a rate of 800 rpm for 24 h to complete the esterification reaction, and add an aqueous sodium hydroxide solution every 2 h during the reaction to adjust the pH of the reaction system to always remain at 8-9; mix the solution obtained after the reaction with ethanol in a ratio of 1:20 (v / v), let it stand for 30 min to obtain a precipitate, discard the supernatant and collect the precipitate, transfer it to a dialysis bag (14 kDa), dialyze it in deionized water for 14 d, and change the dialysis solution every 12 h to completely remove the reaction residues; place the final product obtained after dialysis at -80 °C and freeze-dry it for 7 d to obtain methacrylated carboxymethyl cellulose (SCMA), and store it in a -20 °C desiccator;

[0051] (3) Take the prepared methacrylated carboxymethyl cellulose and dissolve it in a calcium- and phosphorus-free TBS buffer solution (pH = 7.4) to obtain a 0.5 wt.% methacrylated carboxymethyl cellulose solution; take methacrylamide-modified type I collagen and dissolve it in an acetic acid solution to obtain a 2 mg / mL methacrylamide-modified type I collagen solution; mix the two solutions in an equal volume ratio, then add 1.5 wt.% lithium phenyl(2,4,6-trimethylformyl)phosphate, and use a 0.1 M aqueous sodium hydroxide solution to adjust the pH to 8.4 to obtain a gel precursor solution;

[0052] (4) Place the obtained gel precursor solution in an environment at 37 °C and 100% relative humidity and let it stand for 5 min to complete thermosensitive self-assembly; then irradiate it with a blue light source with a wavelength of 405 nm for 1 min to complete photocrosslinking to obtain a biomimetic self-mineralized hydrogel (CS).

[0053] The methacrylated carboxymethyl cellulose (SCMA) prepared in this example was subjected to 1 1H nuclear magnetic resonance detection and infrared detection with the obtained spectra as follows Figure 1 and2 As shown. From Figure 1 it can be seen that the peaks at about 5.7 ppm and 6.1 ppm are the characteristic peaks of the double bonds of the methacrylate group, and the peak at about 2 ppm is the characteristic peak of the methyl group of the methacrylate group, confirming that the methacrylate root has been successfully connected to the carboxymethyl cellulose polymer through an esterification reaction. As Figure 2 shown, the peaks at about 812 cm -1 , 945 cm -1 , 1723 cm -1 are the characteristic peaks of the double bonds of the methacrylate group, confirming that the methacrylate root has been successfully connected to the carboxymethyl cellulose polymer through an esterification reaction.

[0054] The physical pictures of the thermosensitive self-assembly and photocrosslinking processes of the photocrosslinked biomimetic self-mineralized hydrogel obtained by the above method are as Figure 3 shown. From Figure 3 the change of [], it can be seen that the gel precursor solution presents a clear flowing liquid state. After the self-assembly process at 37 °C, a non-flowing turbid gel is formed. Then, after crosslinking by blue light irradiation, a biomimetic self-mineralized hydrogel is formed.

[0055] The rheological test results of the biomimetic self-mineralized hydrogel of this example are as Figure 4 shown. The gel precursor solution is placed on the variable-temperature rheological test sample stage. As the temperature rises from 4 °C to 37 °C, the gel precursor first undergoes a self-assembly process, and the storage modulus shows the first sudden increase. Subsequently, the gel is irradiated with blue light, and the gel undergoes photocrosslinking, and the storage modulus shows the second sudden increase.

[0056] The obtained biomimetic self-mineralized hydrogel is placed in a simulated body fluid (supersaturated calcium phosphate solution). The collagen fibers in the gel can spontaneously recruit calcium and phosphorus for in-situ deposition under the induction of methacrylated carboxymethyl cellulose to achieve intrafibrillar mineralization of collagen. The transmission electron microscope image of the mineralized gel is as Figure 5 shown. It can be seen that obvious nano-hydroxyapatite crystals are formed inside the collagen fibers, and the mineralized crystals are arranged along the long axis of the collagen, forming a nano-hybrid structure similar to bone tissue.

[0057] Example 2

[0058] The preparation method of the biomimetic self-mineralized hydrogel is as follows:

[0059] (1) Dissolve 2 g of carboxymethyl cellulose in deionized water and stir overnight at room temperature to obtain a 2 wt.% aqueous solution of carboxymethyl cellulose; under an ice-water bath, continuously stir the solution at a rate of 800 rpm, take 34 g of methyl methacrylic anhydride, and dropwise add it to the aqueous solution of carboxymethyl cellulose drop by drop under light-shielding conditions to obtain a pre-reaction solution;

[0060] (2) The pre-reaction solution was placed at 4 °C, and the pH of the pre-reaction solution was adjusted to 8 - 9 using 5 M aqueous sodium hydroxide solution. It was continuously stirred at a rate of 800 rpm for 24 h to complete the esterification reaction. During the reaction, aqueous sodium hydroxide solution was added every 2 h to adjust the pH of the reaction system to always remain at 8 - 9. The solution obtained after the reaction was mixed with ethanol at a ratio of 1:20 (v / v), allowed to stand for 30 min to obtain a precipitate. The supernatant was discarded and the precipitate was collected, transferred to a dialysis bag (14 kDa), and dialyzed in deionized water for 14 d, with the dialysis solution being changed every 12 h to thoroughly remove the reaction residues. The final product obtained after dialysis was placed at -80 °C and freeze-dried for 7 d to obtain methacrylated carboxymethyl cellulose, which was stored in a -20 °C desiccator.

[0061] (3) Take the prepared methacrylated carboxymethyl cellulose and dissolve it in freshly prepared TBS buffer containing 9 mM Ca 2+ , 4.2 mM PO 4 3- (pH = 7.4) to obtain a 0.5 wt.% methacrylated carboxymethyl cellulose solution. Take methacrylamide-modified type I collagen and dissolve it in acetic acid solution to obtain a 2 mg / mL methacrylamide-modified type I collagen solution. The two solutions were mixed in equal volume ratio, and then 1.5 wt.% lithium phenyl(2,4,6-trimethylformyl)phosphate was added, and the pH was adjusted to 8.4 using 0.1 M aqueous sodium hydroxide solution to obtain a gel precursor solution.

[0062] (4) The obtained gel precursor solution was placed at 37 °C in an environment with 100% relative humidity and allowed to stand for 5 min to complete the temperature-sensitive self-assembly. Subsequently, it was irradiated with a blue light source with a wavelength of 405 nm for 1 min to complete the photocrosslinking, obtaining a biomimetic self-mineralized hydrogel (CS-ACP).

[0063] The methacrylated carboxymethyl cellulose solution of this example was observed using a cryo-transmission electron microscope. As Figure 6 shown, methacrylated carboxymethyl cellulose assembled into nanoclusters with a diameter of about 30 - 50 nm in the buffer solution containing calcium and phosphorus. The selected area diffraction results showed that the clusters still maintained an amorphous state of the amorphous phase.

[0064] The methacrylated carboxymethyl cellulose solution was further observed using a high-angle annular dark-field scanning transmission electron microscope and energy-dispersive X-ray spectroscopy. The results were as Figure 7 shown. The clusters formed by methacrylated carboxymethyl cellulose in the buffer solution containing calcium and phosphorus contained calcium and phosphorus elements, confirming that methacrylated carboxymethyl cellulose could spontaneously recruit calcium and phosphorus ions in a supersaturated calcium and phosphorus solution to form amorphous-phase nanoclusters similar to the PILP liquid-phase precursor.

[0065] The bionic self-mineralizing hydrogel of this example was left standing for 3 days at 37 °C in an environment with 100% relative humidity. Due to the use of a buffer solution containing calcium and phosphorus, in-situ self-mineralization occurred within the collagen fibers of the gel, forming mineralized collagen fibers. By observing the gel obtained after standing using a scanning electron microscope, the image is as shown in Figure 8 Figure [Figure number not provided in the original]. In the prepared and formed gel, calcium and phosphorus ions entered the interstitial space within the collagen fibers under the mediation of methacrylated carboxymethyl cellulose and crystallized. Without the assistance of other external forces, in-situ self-mineralization achieved intra-fiber mineralization of collagen, forming a bionic nano-hybrid structure.

[0066] The effects of the two bionic self-mineralizing hydrogels prepared in Example 1 and Example 2 were verified through the following tests:

[0067] Cytotoxicity detection:

[0068] The CCK-8 kit was used to detect the cytotoxicity of the gel materials CS and CS-ACP prepared in Example 1 and Example 2. The specific operation was as follows: Rat bone marrow mesenchymal stem cells were co-cultured with the above gel materials. At fixed time points of 1, 3, and 5 days, the culture medium in the well plates was removed, rinsed 3 times with sterile PBS buffer, and then the prepared CCK-8 working solution was added and left standing in a 37 °C cell culture incubator for 1.5 h. After that, the working solution was aspirated, and the absorbance at 450 nm was measured. The test results are shown in Figure 9 Figure [Figure number not provided in the original]. The rat bone marrow mesenchymal stem cells co-cultured with CS and CS-ACP both showed stable cell proliferation. Among them, CS-ACP, due to containing additional calcium and phosphorus elements, could more significantly promote the proliferation of rat bone marrow mesenchymal stem cells, confirming that the gel materials prepared by the present invention have good biocompatibility and can effectively promote stem cell proliferation.

[0069] Effect on the cytoskeleton and spreading morphology:

[0070] To further illustrate the biocompatibility of the gel material, cytoskeleton staining was used to observe the cytoskeleton and morphology of rat bone marrow mesenchymal stem cells after direct contact co-culture with the gel. The specific operation was as follows: Rat bone marrow mesenchymal stem cells were respectively inoculated on the gel materials CS and CS-ACP prepared in Example 1 and Example 2. After culturing for 1 day, they were fixed with 4% paraformaldehyde solution for 15 min, rinsed 3 times with phosphate buffer solution (PBS), permeabilized with 0.5% Triton X-100 solution for 10 min, rinsed 3 times with phosphate buffer solution, incubated with the prepared fluorescein isothiocyanate (FITC)-conjugated phalloidin solution in the dark for 30 min. After the staining was completed, they were rinsed 3 times with phosphate buffer solution and then observed under a confocal microscope. The results are shown in Figure 10As shown, rat bone marrow mesenchymal stem cells can successfully adhere to the surfaces of CS and CS-ACP materials and have good extension, indicating that the materials have good biocompatibility.

[0071] Osteogenic promotion effect:

[0072] To verify that the material has an osteogenic promotion effect, real-time fluorescence quantitative PCR experiments were performed after co-culturing rat bone marrow mesenchymal stem cells in direct contact with the gel to observe the osteogenic promotion effect of the material. The specific operations are as follows: Rat bone marrow mesenchymal stem cells were respectively inoculated on the gel materials CS and CS-ACP prepared in Example 1 and Example 2. After 1 day, the medium was changed to osteogenic induction medium and cultured for 7 days, then RNA was extracted, and reverse transcription and real-time quantitative PCR were performed. The expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference to evaluate the expression level of the osteogenic specific gene alkaline phosphatase (ALP). The results are as Figure 11 shown. Compared with the blank control group, the expression level of alkaline phosphatase in the CS and CS-ACP groups was significantly up-regulated, indicating that the material has the effect of promoting osteogenesis.

[0073] The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those of ordinary skill in the art can make many modifications and variations based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by those skilled in the art in the technical field of the present invention based on the concept of the present invention through logical analysis, reasoning or limited experiments should be within the protection scope determined by the claims.

Claims

1. A method for preparing a biomimetic self-mineralizing hydrogel, characterized in that, it comprises the following steps: (1) Adding methacrylic anhydride to an aqueous solution of carboxymethyl cellulose, and mixing to obtain a pre-reaction solution; (2) Performing an esterification reaction on the pre-reaction solution, and purifying and drying the obtained crude product to obtain methacrylated carboxymethyl cellulose; (3) Dissolving the methacrylated carboxymethyl cellulose in a buffer solution, mixing it with a supersaturated calcium ion and phosphate ion solution, further mixing the obtained mixed solution with a methacrylamide-modified type I collagen solution and a photoinitiator, and adjusting the pH to be alkaline to obtain a gel precursor solution; (4) Subjecting the gel precursor solution to thermosensitive self-assembly and photo-crosslinking to obtain a biomimetic self-mineralizing hydrogel; In the step (1), the molar ratio of the monomer of carboxymethyl cellulose to methacrylic anhydride is 1:1 to 100; In the step (2), the esterification reaction is carried out in an environment with pH = 8 to 9, the temperature of the esterification reaction is 0 to 4 °C, and the reaction time is 4 to 24 h; In the step (3), in the supersaturated calcium ion and phosphate ion solution, the concentration of calcium ions is 4.5 mM to 9 M, the molar ratio of calcium element to phosphorus element is 15:7 to 9, and the pH of the gel precursor solution is 8 to 9; by mass percentage, in the gel precursor solution, the content of methacrylamide-modified type I collagen is 0.1% to 10%, the content of methacrylated carboxymethyl cellulose is 0.25% to 5%, and the content of the photoinitiator is 0.15% to 1%.

2. The method according to claim 1, characterized in that: In the step (3), the pH of the buffer solution is consistent with that of human blood, and its type includes one of TBS buffer solution, MES buffer solution, and HEPES buffer solution.

3. The method according to claim 1, characterized in that: In the step (4), the method of thermosensitive self-assembly is as follows: The gel precursor solution is left standing in an environment with a temperature of 35 to 38 °C and a relative humidity of 90% to 100% until self-assembly is completed.

4. A biomimetic self-mineralizing hydrogel, characterized in that: It is prepared by using the method according to any one of claims 1 to 3.

5. An application of the biomimetic self-mineralizing hydrogel according to claim 4, characterized in that: Its application as a raw material in the preparation of a biological repair material.

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