Bone repair implant based on chill resin and calcium glycerophosphate composite gel and preparation method of bone repair implant

Abstract

The invention belongs to the technical field of bone repair materials, and particularly relates to a bone repair implant based on chill resin and calcium glycerophosphate composite gel and a preparation method of the bone repair implant. According to the bone repair implant based on the cold-bonded resin and calcium glycerophosphate composite gel, cold-bonded resin is selected as a main component of the gel, hyaluronic acid is selected as an auxiliary component, and calcium glycerophosphate, citrate, calcium saccharate and calcium amino acid are used as gel solidification accelerators; the bone repair implant with high elasticity, high toughness and certain strength is formed, it is proved that the bone repair implant can be used as a basic material for cartilage tissue repair, and an effective choice is provided for repair of articular cartilage and other cartilage tissues.

Description

Technical Field

[0001] The invention belongs to the technical field of bone repair materials, and in particular relates to a bone repair implant based on cold-setting resin calcium glycerophosphate composite gel and a preparation method thereof. Background Art

[0002] The human knee joint has a complex structure and function, and is loaded with weight all day long, making it susceptible to various acute injuries and chronic strains. One problem with knee injuries is that compared to bones, articular cartilage damage is much more difficult to recover. After fixation, broken bones can usually heal gradually, but the cartilage tissue on the joint has poor regeneration ability. After wear and tear, it is difficult to expect them to recover to their original state by their own repair ability. The main reasons are: 1. Lack of blood vessels prevents undifferentiated cells from entering the injured area to carry out a healing process similar to other tissues; 2. The cartilage tissue itself lacks undifferentiated cells that can repair the injury; 3. Chondrocytes are embedded in a dense collagen polysaccharide matrix, which limits the proliferation and migration ability of their own cells; 4. Although the synthesis capacity of mature chondrocyte matrix increases when the cartilage is damaged, its limited function is not enough to meet the needs of repair.

[0003] The treatment methods for articular cartilage injuries mainly include: 1. General treatment. General treatment mainly includes muscle function exercises to stabilize the joints, joint braces or protectors to protect the joints, reducing the range of joint movement, and injecting lubricants into the joint cavity. 2. Drug treatment. Drug treatment mainly selects non-steroidal anti-inflammatory drugs and drugs that have the effects of nourishing the liver and kidneys and promoting blood circulation and unblocking collaterals. Drugs that nourish the liver and kidneys and promote blood circulation and unblocking collaterals have a certain auxiliary effect on cartilage repair. 3. Surgical treatment. Surgical treatment mainly includes limited cartilage resection plus drilling, bone realignment surgery to correct force line, artificial joint replacement, cartilage transplantation, and bone resection. 4. Other treatments. Other treatments mainly include wax therapy and ultrashort wave therapy.

[0004] None of the above treatments can truly repair cartilage tissue and it is difficult to restore full function.

[0005] Therefore, the repair of cartilage, especially articular cartilage, has always troubled patients and clinicians. Now, many researchers are exploring ways to repair articular cartilage damage, such as using stem cells to promote tissue repair, but these explorations have not been successful enough. At present, protecting the knees and preventing injuries are still the best solutions. Controlling weight, engaging in reasonable physical activities and exercising lower limb strength can help people reduce the strain on the knee cartilage.

[0006] Since Chestman and Smith first began to study the in vitro culture of chondrocytes for cartilage defect repair in 1965, people have begun to have a new understanding of the concept that articular cartilage damage cannot be repaired by cartilage proliferation. Experiments have shown that not only young but also old articular cartilage specimens can still culture new hyaline cartilage in vitro. Although the proliferation capacity of chondrocytes is limited, their quality and quantity directly affect the effect of in vitro culture and expansion, and the biological characteristics of chondrocytes in different growth environments are also different. Chondrocytes grow well in suspension culture or semi-solid agar culture medium and maintain a stable phenotype. When cultured for a long time in a four-dimensional culture environment covered with hydrogel at the bottom of the culture bottle, chondrocytes can form nodular structures. Their cell morphology, extracellular matrix secretion and cartilage-specific gene expression are similar to articular cartilage.

[0007] In recent years, with the deepening of research on bone repair materials, it has gradually expanded from the regeneration and repair of bone tissue to the repair of cartilage tissue, and attempts have been made to use biomaterials to repair damaged cartilage tissue and restore its function. Among them, hyaluronic acid is injected into the joints as a lubricant, glucosamine and chondroitin sulfate are used for drug treatment, and stem cells and materials are mixed for treatment. More than hundreds of thousands of papers have been published on these attempts, but materials that can be used for clinical treatment and cartilage repair are very rare. This also shows that the core problem that plagues cartilage repair, that is, the key basic materials have not been found, and the mechanism of synergy between materials and stem cells has not been fully understood. Therefore, it is necessary to find more effective materials as the basic materials for cartilage repair.

[0008] Gellan gum is a polymer of pyranoglucopyranose, 6-deoxy-L-pyranose and D-pyranose acetate, calcium potassium sodium salt, also known as gellan gum, CAS No. 71010-52-1. It is colorless, transparent and highly tough. It is the main component for preparing plant tissue culture medium and microbial culture medium. It is a substitute for agar secreted by Pseudomonas and is colorless, transparent and highly tough.

[0009] Sodium hyaluronate is a physiologically active substance widely present in the human body. It is a high molecular weight linear mucopolysaccharide composed of disaccharide units composed of glucuronic acid and acetylhexosamine, with a molecular weight of 1 million. It forms a viscous and elastic solution in water with physiological pH and ionic strength. Sodium hyaluronate is the main component of synovial fluid and one of the components of cartilage matrix. It plays a lubricating role in the joint cavity, which can reduce friction between tissues. At the same time, it plays its elastic role, buffers the effect of stress on articular cartilage, and plays its due physiological function.

[0010] Calcium glycerophosphate is a white crystalline powder. It is odorless, almost odorless, slightly bitter, and hygroscopic. It has the function of improving tissue cells and forming bone tissue, while also playing a role in the metabolism of calcium glycerophosphate salts and replenishing brain vitality.

[0011] Citrate, calcium saccharate and amino acid calcium are all osteogenic components that are more easily absorbed and utilized by tissues than inorganic calcium and participate in metabolic cycles. Summary of the invention

[0012] Based on the above background, the present invention designs a bone repair implant based on composite gel, selects plant gel, i.e. cold-setting resin, as the main component of the gel, selects sodium hyaluronate as the auxiliary component, and uses calcium glycerophosphate and citrate, calcium saccharate and amino acid calcium as gel coagulation promoters to form a bone repair implant with high elasticity, high toughness and certain strength, and attempts to use it as a basic material for cartilage tissue repair, providing an effective choice for the repair of articular cartilage and other cartilage tissues.

[0013] Specifically, in one aspect, the present invention provides a bone repair implant based on a cold-setting resin-calcium glycerophosphate composite gel, which comprises components A, B and C, wherein component A is a cold-setting resin, component B is sodium hyaluronate, and component C is calcium glycerophosphate, magnesium citrate, calcium saccharate and amino acid calcium, wherein components A, B and C form a composite gel.

[0014] Furthermore, the sodium hyaluronate includes high molecular weight sodium hyaluronate and medium molecular weight sodium hyaluronate.

[0015] Furthermore, the molecular weight range of the high molecular weight sodium hyaluronate is 1,800,000 to 2,200,000, and the molecular weight range of the medium molecular weight sodium hyaluronate is 1,000,000 to 1,800,000.

[0016] Furthermore, the mass ratio of the high molecular weight sodium hyaluronate to the medium molecular weight sodium hyaluronate is 0.5-2:2-0.5.

[0017] Furthermore, the calcium glycerophosphate includes one or more of β-calcium glycerophosphate, D(+)α-calcium glycerophosphate and L(-)α-calcium glycerophosphate.

[0018] Furthermore, the calcium saccharate includes one or both of calcium gluconate and calcium L-threonate.

[0019] Furthermore, the amino acid calcium includes one or more of calcium glycinate, calcium lysine, calcium L-aspartate, calcium aspartate, calcium glutamate and calcium L-histidine.

[0020] In another aspect, the present invention provides a method for preparing a bone repair implant based on a cold-setting resin calcium glycerophosphate composite gel as described herein, which comprises separately preparing aqueous solutions of components A, B and C, first mixing the aqueous solutions of components A and B, then adding the aqueous solution of component C, stirring and mixing, and then standing to form a gel, and then freeze-drying to obtain the bone repair implant based on the cold-setting resin calcium glycerophosphate composite gel.

[0021] Further, the aqueous solutions of component A, component B and component C are mixed in a volume ratio of 1.0-2.0:1.0-2.0:0.5-2.5.

[0022] Further, the aqueous solution of component A is prepared as follows: dissolving cold-bonding resin in water that has passed endotoxin testing at a concentration of 1.0-20 g / L to obtain a clear and transparent solution for use; further, wherein the dissolution is carried out at 0-80° C. under ultrasound or stirring.

[0023] Further, the aqueous solution of component B is prepared as follows: sodium hyaluronate is dissolved in water that has passed endotoxin testing at a concentration of 5.0-20.0 g / L to obtain a clear and transparent solution for use; further, the dissolution is carried out at 0-80° C. under ultrasound or stirring.

[0024] Furthermore, the aqueous solution of component C is prepared as follows: calcium glycerophosphate is dissolved in water that has passed the endotoxin test at a concentration of 10.0-25.0 g / L to obtain a clear and transparent solution I; magnesium citrate, calcium saccharate and amino acid calcium are dissolved in water that has passed the endotoxin test at a concentration of 5.0-25.0 g / 100 ml to obtain a clear and transparent solution II; solution I and solution II are mixed in a volume ratio of 1.0:0.5-2.5 to obtain an aqueous solution of component C and stored below 10°C for standby use.

[0025] Furthermore, the mass ratio of magnesium citrate, calcium saccharate and amino acid calcium is 10-25:20-50:70-150.

[0026] Further, the dissolution of calcium glycerophosphate is carried out below 20° C. under ultrasound or stirring.

[0027] Furthermore, the dissolution of magnesium citrate, calcium saccharate and calcium amino acid is carried out at room temperature under ultrasound or stirring.

[0028] Furthermore, the step of standing to form a gel comprises first heating the mixed aqueous solution of components A, B and C to 50-80° C. under nitrogen protection, maintaining the temperature for 15-45 minutes, and then transferring the mixed aqueous solution to a mold, cooling the mixed aqueous solution to room temperature, and maintaining the temperature for 2-10 hours.

[0029] Furthermore, the preparation method also includes slicing or cutting the formed gel before freeze-drying to obtain a desired shape and size.

[0030] In other aspects, the present invention also provides the use of a bone repair implant based on a cold-setting resin calcium glycerophosphate composite gel as described herein or prepared by the method described herein in cancellous bone and / or cartilage repair.

[0031] Advantageous Effects of the Invention

[0032] The present invention designs a bone repair implant based on cold-setting resin calcium glycerophosphate composite gel, selects cold-setting resin as the main component of the gel, selects hyaluronic acid as the auxiliary component, and uses calcium glycerophosphate and citrate, calcium saccharate and amino acid calcium as gel solidification accelerators to form a bone repair implant with high elasticity, high toughness and certain strength, and it is confirmed that it can be used as a basic material for cartilage tissue repair, providing an effective choice for the repair of articular cartilage and other cartilage tissues. DETAILED DESCRIPTION

[0033] The present invention relates to a bone repair implant based on cold-setting resin calcium glycerophosphate composite gel and a preparation method thereof, and is particularly related to the repair and functional recovery of cancellous bone and cartilage tissue. According to the composition, structure and performance characteristics of cancellous bone and cartilage tissue, cold-setting resin is selected as one of the main core components of the gel, hyaluronic acid is selected as another core component, and calcium glycerophosphate, citrate, calcium saccharate and amino acid calcium are used as gel solidification accelerators to form a bone repair implant with high elasticity, high toughness and certain strength.

[0034] In order to obtain a bone repair implant based on cold-setting resin calcium glycerophosphate composite gel, the present invention provides the following specific implementation technical solutions.

[0035] First, cold-setting resin is selected as the main component of the gel, and tested water (the water used in the present invention, such as deionized water and distilled water, has passed endotoxin testing to ensure that the water used is sterile, non-toxic and safe. For example, the water to be used can be calibrated for endotoxin using a colorimetric method) is used to prepare a cold-setting resin solution (I) with a concentration of 1.0-20 g / L, preferably 1.5-10 g / L.

[0036] Specifically, use cold-bonded resin with a purity of ≥99%, accurately weigh 1.0-20g (accurate to 0.01g) of cold-bonded resin, add it to a 1L volumetric flask, then add 600ml of water that has been tested for endotoxin, ultrasonically centrifuge in 0-80℃ water for 10-30 minutes or stir until all of it is dissolved, then add water to the 1L mark, ultrasonically centrifuge for 3-5 minutes or stir, and set aside the formed clear and transparent solution.

[0037] Furthermore, sodium hyaluronate is selected as another core component. The structure of sodium hyaluronate is shown below:

[0038]

[0039] Sodium hyaluronate is a high molecular weight carbohydrate compound with different molecular weights of unit repetition. It is white or off-white granules or powders, odorless, and has a nitrogen content of 2.8%-4.0% and a glucuronic acid content of 37.0%-51.0% when dry. Hyaluronic acid includes macromolecular hyaluronic acid (molecular weight range 1800000-2200000); medium molecular hyaluronic acid (molecular weight range 1000000-1800000); small molecular hyaluronic acid (molecular weight range 400000-1000000). The size of the molecular weight has an important influence on its viscosity, moisturizing and other properties. Therefore, the performance of the gel obtained by selecting different molecular weight sodium hyaluronates is very different. Since the repair of cartilage and cancellous bone has a long period, the gel needs to maintain its shape for a relatively long time. Therefore, the present invention selects macromolecular sodium hyaluronate and medium molecular weight sodium hyaluronate as components of the gel.

[0040] The tested water is used to prepare an aqueous solution of sodium hyaluronate (II) composed of high molecular weight sodium hyaluronate and medium molecular weight sodium hyaluronate, the concentration of which is 5.0-20.0 g / L, preferably 10-15 g / L, wherein the mass ratio of high molecular weight sodium hyaluronate to medium molecular weight sodium hyaluronate is 0.5-2:2-0.5.

[0041] Specifically, in a clean room, accurately weigh high molecular weight sodium hyaluronate, such as 10g, and medium molecular weight sodium hyaluronate, such as 5g. Add the weighed high molecular weight and medium molecular weight sodium hyaluronate into a 1L volume, then add 600ml of water tested for endotoxin, ultrasonically centrifuge in 0-80℃ water for 10-30 minutes or stir until all are dissolved, then add water to the 1L mark, ultrasonically centrifuge for 3-5 minutes or stir, and set aside the formed clear and transparent solution.

[0042] Further, the present invention selects calcium glycerophosphate As a gel formation promoter. Calcium glycerophosphate exists in three isomers: β-glycerophosphate calcium ((HOCH2)2CHOPO3Ca), D(+) and L(-)α-glycerophosphate calcium (HOCH2CH(OH)CH2OPO3Ca), and one or more of them can be used simultaneously. The present invention uses it as a calcium ion donor to accelerate the gelation process, and at the same time as a calcium and phosphate donor for bone tissue formation. The present invention prepares it into an aqueous solution for use, and the concentration of the aqueous solution is 10.0-25.0g / L. Specifically, in a clean workshop, accurately weigh 10.0-25.0g of calcium glycerophosphate, add it to a 1L volumetric flask, and then add 600ml of water that has been tested for endotoxins and ultrasonically centrifuge it in water below 20°C for 10-30 minutes or stir until it is completely dissolved, then add water to the 1L scale, and then ultrasonically centrifuge it for 5-30 minutes or stir it, and the formed clear and transparent solution is set aside. The preparation temperature of the aqueous solution of calcium glycerophosphate is below 20°C, and the storage temperature of the aqueous solution is not higher than 10°C.

[0043] Also choose magnesium citrate Calcium Gluconate Calcium L-Threonate Calcium and magnesium salts with higher solubility such as amino acid calcium are used as gel formation promoters and regulators of calcium-phosphorus ratio. The amino acid calcium that can be used is preferably soluble in water and has a higher solubility in water, such as calcium glycinate. Calcium Lysinate Calcium L-Aspartate Calcium Aspartate Calcium Glutamate Calcium L-histidine etc., but not limited to the above amino acid calcium.

[0044] The present invention prepares it into an aqueous solution for use, and the concentration is 5.0-25.0g / 100ml. Specifically, in a clean workshop, accurately weigh 50.0-250.0g of one or more of magnesium citrate, calcium gluconate, calcium L-threonate and amino acid calcium, add them to a 1L volumetric flask, then add 600ml of water tested for endotoxin, ultrasonically centrifuge in normal temperature water for 5-30 minutes, completely dissolve, then add water to the 1L scale, ultrasonically centrifuge for 5-30 minutes, and use the formed clear and transparent solution for standby use.

[0045] Further, the aqueous solution of magnesium citrate, calcium saccharate and amino acid calcium is mixed with the aqueous solution of calcium glycerophosphate to form a composite aqueous solution (III) of magnesium citrate, calcium saccharate and amino acid calcium aqueous solution and calcium glycerophosphate aqueous solution enriched with calcium, phosphorus, sugar and amino acid components. Considering the co-solubility of various anions and cations after mixing and the effective content of calcium, phosphorus, sugar and amino acid components in the solution. The ratio of the aqueous solution of magnesium citrate, calcium saccharate and amino acid calcium to the aqueous solution of calcium glycerophosphate is (V) 0.5-2.5:1.0. Within this ratio range, a high concentration of anion and cation coexistence solution can be formed without precipitation, providing a favorable chemical and physical environment for gel preparation.

[0046] Further, the cold resin solution (I), sodium hyaluronate solution (II) and the composite solution (III) formed by magnesium citrate, calcium saccharate and amino acid calcium solution and glycerophosphate calcium solution are sterilized with ethylene oxide, (I), (II) and (III) are mixed according to the volume ratio (V) of 1.0-2.0:1.0-2.0:0.5-2.5, specifically, (I) is added to (II) under stirring, and then (III) is added to the mixed solution of (I) and (II), stirred for 5-10 minutes, and then allowed to stand to form a gel. The temperature of (I), (II) and (III) before addition is preferably not higher than 10°C, and after mixing, the temperature is slowly raised to 60°C under nitrogen protection, maintained for 15-45 minutes, and then transferred to a circular, rectangular and cube mold, cooled to room temperature, maintained for 2-10 hours, and then the gel is taken out and sliced ​​or cut to obtain the required shape and size.

[0047] Furthermore, the partially sliced ​​or cut gel is freeze-dried to obtain a bone repair implant based on a porous cold-hardening resin calcium glycerophosphate composite gel. Conventional drying causes the gel to shrink, form wrinkles, and destroy the gel structure, but during the freeze-drying process, the structure of the sample will not be destroyed because the solid components are supported by the solid ice in their position. When the ice sublimates, pores are left in the dried remaining material. This preserves the integrity of the product's biological and chemical structure and its activity. Therefore, the present invention uses a freeze-drying method to dry the formed cold-hardening resin calcium glycerophosphate composite gel bone repair implant to form a porous gel material, which provides space for the growth of blood vessels and enhances the physiological activity of the cold-hardening resin calcium glycerophosphate composite gel bone repair implant.

[0048] Furthermore, the obtained cold-setting resin calcium glycerophosphate composite gel was subjected to performance tests:

[0049] (1) Compression test method: Load speed 1 mm / min, compression strength: Prepare a sample (diameter 6 mm, height 12 mm), and use a universal mechanical testing instrument to measure the mechanical strength of the sample. Each sample shall be measured at least 5 times.

[0050] (2) Degradation test method: Take the injection molded material and conduct degradation test in PBS solution: The preparation method of PBS is as follows: accurately weigh KH2PO4 (0.544g), Na2HPO4·12H2O (7.16g), NaCl (16g) and KCl (0.402g) and dissolve them in 2L deionized water, and use a volumetric flask to make up the volume. The pH measurement range is between 7.2-7.4; put the sample into a centrifuge tube and add a certain amount of PBS solution. The volume ratio of PBS solution to sample mass is 1g / 30mL; then place the centrifuge tube containing the sample in a constant temperature oscillating box at a temperature of 37℃ and a speed of 80rpm / min; take out the sample at 1 (1D), 1w, 2w, 3w and 4w, weigh it and calculate the weight loss rate. Set up three control groups for each sample.

[0051] (3) pH determination: The supernatant of the sample was measured using a pH meter and PBS was replaced every 7 days.

[0052] (4) Cytotoxicity and cell proliferation rate test: Prepare the extraction solution in accordance with the provisions of T16886 on biological materials, and calculate the proliferation rate by comparing the standard extract with the blank.

[0053] (5) Cultivation and growth of bone cells in gel extract.

[0054] The prepared cold-gel resin calcium glycerophosphate composite gel was immersed in Dulbecco's modified Eagle's medium (DMEM), and the sample extract (200 mg / mL) was prepared according to ISO 10993-12. Mouse bone marrow mesenchymal stem cells (rBMSCs; ATCC, BeNa Culture Collection, Beijing, China) were cultured at 1×10 3 The cells were seeded in 96-well plates at a density of 10 cells / well. On days 1, 3, and 5, the viability of rBMSCs cells was quantified by CCK-8 assay, and the absorbance was read at 450 nm by a microplate reader. After staining with rhodamine-labeled phalloidin (FITC) and 4,6-diamidino-2-phenylindole (DAPI), the cell morphology was observed using an inverted fluorescence microscope. Alkaline phosphatase (ALP) activity was used to detect early osteogenic differentiation. rBMSCs cells were cultured at 5×10 4 The cells were seeded at a density of 10 cells / well in a 24-well plate and cultured with an extract supplemented with osteogenic differentiation medium (0.05 mmol / L vitamin C, 10 mmol / L β-glycerophosphate, and 0.1 μmol / L dexamethasone). After 7 and 11 days, the cells were cultured according to the protocol of the ALP kit. The final ALP activity was normalized to protein concentration using a BCA kit. Alizarin red staining was used to determine cell mineralization in the late stage of differentiation. rBMSCs cells were cultured at 5×10 4Cells were seeded at a density of 10 cells / well in 24-well plates. After 7 and 14 days, cells were rinsed with PBS, fixed with 4% paraformaldehyde for 20 min, and stained with Alizarin Red for 20 min at room temperature. The stained nodules were incubated in 10% cetylpyridinium chloride for 30 min and measured by a microplate reader at 620 nm.

[0055] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0056] Example 1

[0057] (1) Preparation of cold-bonding resin solution: Accurately weigh 10.0 g of cold-bonding resin and add it to a 1-liter volumetric flask. Then add 600 ml of endotoxin-tested water. Ultrasonic centrifuge in 20°C water for 15 minutes to dissolve it completely. Then add water to the 1-liter mark and ultrasonicate for another 5 minutes. The resulting clear and transparent solution will be set aside for later use.

[0058] (2) Sodium hyaluronate solution (II): Accurately weigh 10.0 g of high molecular weight sodium hyaluronate (molecular weight range: 1,800,000-2,200,000) and 5.0 g of medium molecular weight sodium hyaluronate (molecular weight range: 1,000,000-1,800,000) and add them to a 1 L container. Then add 600 ml of endotoxin-tested water and perform ultrasonic centrifugation in 20°C water for 15 minutes to completely dissolve the hyaluronate. Then add water to the 1 L mark and perform ultrasonication for another 5 minutes. The resulting clear and transparent solution will be set aside for use.

[0059] (3) Composite solution (III) formed by magnesium citrate, calcium saccharate and amino acid calcium solution and glycerophosphate calcium solution: accurately weigh 15 g of β-glycerophosphate calcium and add it to a 1L volumetric flask, then add 600 ml of water tested for endotoxin, ultrasonically centrifuge in 20°C water for 15 minutes until all the solution is dissolved, then add water to the 1L mark, ultrasonically centrifuge for 10 minutes, and set aside the clear and transparent solution formed. Store at below 10°C for later use.

[0060] Accurately weigh 10.0g of magnesium citrate, 20.0g of calcium gluconate, 30.0g of calcium glycinate, 30.0g of calcium lysine, and 10.0g of calcium phenylalanine, add to a 1L volumetric flask, then add 600ml of endotoxin-tested water, ultrasonically centrifuge in room temperature water for 10 minutes to dissolve all, then add water to the 1L mark, ultrasonicate for 10 minutes, and set aside the formed clear and transparent solution.

[0061] 400 ml of β-glycerophosphate calcium aqueous solution and 200 ml of magnesium citrate, calcium gluconate and amino acid calcium aqueous solution were measured and mixed, and ultrasonicated for 5 minutes to form a composite aqueous solution of magnesium citrate, calcium gluconate and amino acid calcium aqueous solution and glycerophosphate calcium (III).

[0062] (4) Preparation of cold-setting resin calcium glycerophosphate composite gel: (I), (II) and (III) are sterilized with ethylene oxide. 150 ml (I), 150 ml (II) and 200 ml (III) are measured, (I) is added to (II), and then (III) is added to the mixture of (I) and (II), and stirred and ultrasonicated for 3 minutes. The temperature is slowly raised to 60°C under nitrogen protection and maintained for 15 minutes, and then transferred to round, rectangular and cube molds and maintained for 5 hours, and then the gel is taken out and sliced ​​or cut.

[0063] (5) Preparation of porous cold-setting resin calcium glycerophosphate composite gel: freeze-dry the sliced ​​or cut gel from part (4) to obtain a porous cold-setting resin calcium glycerophosphate composite gel bone repair implant.

[0064] (6) Performance test of cold-setting resin calcium glycerophosphate composite gel. According to the test contents of the above implementation scheme, (1) compression test method; (2) degradation test; (3) pH determination; (4) cytotoxicity and cell proliferation rate test; (5) culture and growth of bone cells in gel.

[0065] Test results:

[0066] (1) Compressive strength and elasticity (MPa,%): 0.75, 36

[0067] (2) Degradation results (%): 1D: 9.5; 1w: 22.3; 2w: 46.1; 3w: 58.9; 4w: 83.2

[0068] (3) pH measurement: 1D: 7.05; 1w: 7.21; 2w: 7.25; 3w: 7.10; 4w: 7.35

[0069] (4) Cytotoxicity and cell proliferation rate (%) test: Level 0; 98 (compared with blank)

[0070] (5) Cultivation and growth status of bone cells in gel leaching solution: Compared with the control sample, it showed better growth status and OD value.

[0071] (6) Gel porosity after freeze drying (%): 78

[0072] Example 2 (same as Example 1, but with a different concentration of cold-bonded resin aqueous solution, increased by 50%)

[0073] (1) Preparation of cold-bonding resin solution: Accurately weigh 15.0 g of cold-bonding resin and add it to a 1-liter volumetric flask. Then add 600 ml of endotoxin-tested water. Ultrasonic centrifuge in 20°C water for 15 minutes to dissolve it completely. Then add water to the 1-liter mark and ultrasonicate for another 5 minutes. The resulting clear and transparent solution will be set aside for later use.

[0074] (2)-(5) are the same as in Example 1.

[0075] Test results:

[0076] (1) Compressive strength and elasticity (MPa,%): 1.05, 45

[0077] (2) Degradation results (%): 1D: 8.2; 1w: 25.1; 2w: 49.4; 3w: 62.7; 4w: 85.3

[0078] (3) pH measurement: 1D: 7.02; 1w: 7.23; 2w: 7.27; 3w: 7.22; 4w: 7.33

[0079] (4) Cytotoxicity and cell proliferation rate (%) test: Level 0; 99 (compared with blank)

[0080] (5) Cultivation and growth status of bone cells in gel leaching solution: Compared with the control sample, it showed better growth status and OD value.

[0081] (6) Gel porosity after freeze drying (%): 70

[0082] Example 3 (same as Example 1, different concentration of sodium hyaluronate solution, but the same ratio of high molecular weight to medium molecular weight)

[0083] (1) Same as Example 1

[0084] (2) Sodium hyaluronate solution (II): Accurately weigh 15.0 g of high molecular weight sodium hyaluronate (molecular weight range: 1,800,000-2,200,000) and 7.50 g of medium molecular weight sodium hyaluronate (molecular weight range: 1,000,000-1,800,000) and add them to a 1 L container. Then add 600 ml of endotoxin-tested water and perform ultrasonic centrifugation in 20°C water for 15 minutes to completely dissolve the hyaluronate. Then add water to the 1 L mark and perform ultrasonication for another 5 minutes. The resulting clear and transparent solution will be set aside for later use.

[0085] (3)-(5) Same as Example 1

[0086] Test results:

[0087] (1) Compressive strength and elasticity (MPa,%): 1.13,43

[0088] (2) Degradation results (%): 1D: 8.3; 1w: 23.9; 2w: 49.5; 3w: 58.9; 4w: 86.1

[0089] (3) pH measurement: 1D: 7.01; 1w: 7.23; 2w: 7.21; 3w: 7.32; 4w: 7.29

[0090] (4) Cytotoxicity and cell proliferation rate (%) test: Level 0; 102 (compared with blank)

[0091] (5) Cultivation and growth status of bone cells in gel leaching solution: Compared with the control sample, it showed better growth status and OD value.

[0092] (6) Gel porosity after freeze drying (%): 76

[0093] Example 4 (same as Example 1, except that the ratio of high molecular weight to medium molecular weight sodium hyaluronate is different and adjusted to 1:1)

[0094] (1) Same as Example 1.

[0095] (2) Sodium hyaluronate solution (II): Accurately weigh 10.0 g of high molecular weight sodium hyaluronate (molecular weight range: 1,800,000-2,200,000) and 10.0 g of medium molecular weight sodium hyaluronate (molecular weight range: 1,000,000-1,800,000) and add them to a 1 L container. Then add 600 ml of endotoxin-tested water and perform ultrasonic centrifugation in 20°C water for 15 minutes to completely dissolve the hyaluronate. Then add water to the 1 L mark and perform ultrasonication for another 5 minutes. The resulting clear and transparent solution will be set aside for later use.

[0096] (3)-(5) are the same as in Example 1.

[0097] Test results:

[0098] (1) Compressive strength and elasticity (MPa,%): 1.21,45

[0099] (2) Degradation results (%): 1D: 9.2; 1w: 23.1; 2w: 46.8; 3w: 59.8; 4w: 83.7

[0100] (3) pH measurement: 1D: 7.03; 1w: 7.26; 2w: 7.31; 3w: 7.25; 4w: 7.30

[0101] (4) Cytotoxicity and cell proliferation rate (%) test: 0 level; 101 (compared with blank)

[0102] (5) Cultivation and growth status of bone cells in gel leaching solution: Compared with the control sample, it showed better growth status and OD value.

[0103] (6) Gel porosity after freeze drying (%): 65

[0104] Example 5 (same as Example 1, different concentrations of calcium glycerophosphate solution)

[0105] (1)-(2) Same as Example 1.

[0106] (3) Composite solution (III) formed by magnesium citrate, calcium saccharate and amino acid calcium solution and glycerophosphate calcium solution: accurately weigh 20.0 g of β-glycerophosphate calcium and add it to a 1L volumetric flask, then add 600 ml of water tested for endotoxin, ultrasonically centrifuge in 20°C water for 15 minutes until all the solution is dissolved, then add water to the 1L mark, ultrasonically centrifuge for 10 minutes, and set aside the clear and transparent solution formed. Store at below 10°C for later use.

[0107] Accurately weigh 10.0g of magnesium citrate, 20.0g of calcium gluconate, 30.0g of calcium glycinate, 30.0g of calcium lysine, and 10.0g of calcium phenylalanine, add to a 1L volumetric flask, then add 600ml of endotoxin-tested water, ultrasonically centrifuge in room temperature water for 10 minutes to dissolve all, then add water to the 1L mark, ultrasonicate for 10 minutes, and set aside the formed clear and transparent solution.

[0108] 400 ml of β-glycerophosphate calcium aqueous solution and 200 ml of magnesium citrate, calcium gluconate and amino acid calcium aqueous solution were measured and mixed, and ultrasonicated for 5 minutes to form a composite aqueous solution of magnesium citrate, calcium gluconate and amino acid calcium aqueous solution and glycerophosphate calcium (III).

[0109] (4)-(5) Same as Example 1.

[0110] Test results:

[0111] (1) Compressive strength and elasticity (MPa,%): 0.94,37

[0112] (2) Degradation results (%): 1D: 8.8; 1w: 21.2; 2w: 44.4; 3w: 56.; 4w: 81.7

[0113] (3) pH measurement: 1D: 7.05; 1w: 7.23; 2w: 7.31; 3w: 7.33; 4w: 7.35

[0114] (4) Cytotoxicity and cell proliferation rate (%) test: Level 0; 103 (compared with blank)

[0115] (5) Cultivation and growth status of bone cells in gel leaching solution: Compared with the control sample, it showed better growth status and OD value.

[0116] (6) Gel porosity after freeze drying (%): 68

[0117] Example 6 (same as Example 1, but with different concentrations of magnesium citrate, calcium gluconate and amino acid calcium aqueous solutions, increased by 20% in the same proportion)

[0118] (1)-(2) Same as Example 1.

[0119] (3) Composite solution (III) formed by magnesium citrate, calcium saccharate and amino acid calcium solution and glycerophosphate calcium solution: accurately weigh 15 g of β-glycerophosphate calcium and add it to a 1L volumetric flask, then add 600 ml of water tested for endotoxin, ultrasonically centrifuge in 20°C water for 15 minutes until all the solution is dissolved, then add water to the 1L mark, ultrasonically centrifuge for 10 minutes, and set aside the clear and transparent solution formed. Store at below 10°C for later use.

[0120] Accurately weigh 12.0g of magnesium citrate, 24.0g of calcium gluconate, 36.0g of calcium glycinate, 36.0g of calcium lysine, and 12.0g of calcium phenylalanine, add to a 1L volumetric flask, then add 600ml of endotoxin-tested water, ultrasonically centrifuge in room temperature water for 10 minutes to dissolve all, then add water to the 1L mark, ultrasonicate for 10 minutes, and set aside the formed clear and transparent solution.

[0121] 400 ml of β-glycerophosphate calcium aqueous solution and 200 ml of magnesium citrate, calcium gluconate and amino acid calcium aqueous solution were measured and mixed, and ultrasonicated for 5 minutes to form a composite aqueous solution of magnesium citrate, calcium gluconate and amino acid calcium aqueous solution and glycerophosphate calcium (III).

[0122] (4)-(5) Same as Example 1.

[0123] Test results:

[0124] (1) Compressive strength and elasticity (MPa,%): 1.33, 49

[0125] (2) Degradation results (%): 1D: 10.8; 1w: 25.9; 2w: 49.7; 3w: 60.3; 4w: 85.3

[0126] (3) pH measurement: 1D: 7.12; 1w: 7.23; 2w: 7.30; 3w: 7.33; 4w: 7.35

[0127] (4) Cytotoxicity and cell proliferation rate (%) test: Level 0; 103 (compared with blank)

[0128] (5) Cultivation and growth status of bone cells in gel leaching solution: Compared with the control sample, it showed better growth status and OD value.

[0129] (6) Gel porosity after freeze drying (%): 71

[0130] Example 7 (same as Example 1, the ratio of magnesium citrate, calcium saccharate and amino acid calcium solution to glycerophosphate calcium solution is different and adjusted to 1:1)

[0131] (1)-(2) Same as Example 1.

[0132] (3) Composite solution (III) formed by magnesium citrate, calcium saccharate and amino acid calcium solution and glycerophosphate calcium solution: accurately weigh 15 g of β-glycerophosphate calcium and add it to a 1L volumetric flask, then add 600 ml of water tested for endotoxin, ultrasonically centrifuge in 20°C water for 15 minutes until all the solution is dissolved, then add water to the 1L mark, ultrasonically centrifuge for 10 minutes, and set aside the clear and transparent solution formed. Store at below 10°C for later use.

[0133] Accurately weigh 10.0g of magnesium citrate, 20.0g of calcium gluconate, 30.0g of calcium glycinate, 30.0g of calcium lysine, and 10.0g of calcium phenylalanine, add to a 1L volumetric flask, then add 600ml of endotoxin-tested water, ultrasonically centrifuge in room temperature water for 10 minutes to dissolve all, then add water to the 1L mark, ultrasonicate for 10 minutes, and set aside the formed clear and transparent solution.

[0134] Measure 200 ml of β-glycerophosphate calcium aqueous solution, 200 ml of magnesium citrate, calcium gluconate and amino acid calcium aqueous solution, mix them and ultrasonicate for 5 minutes to form a composite aqueous solution of magnesium citrate, calcium gluconate and amino acid calcium aqueous solution and glycerophosphate calcium (III).

[0135] (4)-(5) are the same as in the embodiment.

[0136] Test results:

[0137] (1) Compressive strength and elasticity (MPa,%): 1.33, 39

[0138] (2) Degradation results (%): 1D: 8.3; 1w: 20.7; 2w: 45.4; 3w: 59.4; 4w: 82.1

[0139] (3) pH measurement: 1D: 7.15; 1w: 7.29; 2w: 7.32; 3w: 7.32; 4w: 7.36

[0140] (4) Cytotoxicity and cell proliferation rate (%) test: Level 0; 105 (compared with blank)

[0141] (5) Cultivation and growth status of bone cells in gel leaching solution: Compared with the control sample, it showed better growth status and OD value.

[0142] (6) Gel porosity after freeze drying (%): 69

[0143] Example 8 (same as Example 1, the types of magnesium citrate, calcium gluconate and amino acid calcium aqueous solution are different, L-calcium threonate replaces calcium gluconate; L-calcium histidine replaces calcium lysine)

[0144] (1)-(2) Same as Example 1.

[0145] (3) Composite solution (III) formed by magnesium citrate, calcium L-threonate and amino acid calcium solution and glycerophosphate calcium solution: accurately weigh 15 g of β-glycerophosphate calcium and add it to a 1L volumetric flask, then add 600 ml of water tested for endotoxin, ultrasonically centrifuge in 20°C water for 15 minutes to dissolve all the solution, then add water to the 1L mark, ultrasonicate for 10 minutes, and set aside the clear and transparent solution formed. Store at below 10°C for later use.

[0146] Accurately weigh 10.0 g of magnesium citrate, 20.0 g of calcium L-threonate, 30.0 g of calcium glycinate, 30.0 g of calcium L-histidine, and 10.0 g of calcium phenylalanine, add to a 1 L volumetric flask, then add 600 ml of endotoxin-tested water, ultrasonically centrifuge in room temperature water for 10 minutes to dissolve all, then add water to the 1 L mark, ultrasonicate for 10 minutes, and set aside the formed clear and transparent solution.

[0147] 400 ml of β-glycerophosphate calcium aqueous solution and 200 ml of magnesium citrate, calcium gluconate and amino acid calcium aqueous solution were measured and mixed, and ultrasonicated for 5 minutes to form a composite aqueous solution of magnesium citrate, calcium gluconate and amino acid calcium aqueous solution and glycerophosphate calcium (III).

[0148] (4)-(5) Same as Example 1.

[0149] Test results:

[0150] (1) Compressive strength and elasticity (MPa,%): 0.89,35

[0151] (2) Degradation results (%): 1D: 9.2; 1w: 21.7; 2w: 45.3; 3w: 58.3; 4w: 82.6

[0152] (3) pH measurement: 1D: 7.11; 1w: 7.25; 2w: 7.32; 3w: 7.33; 4w: 7.34

[0153] (4) Cytotoxicity and cell proliferation rate (%) test: 0 level; 101 (compared with blank)

[0154] (5) Cultivation and growth status of bone cells in gel leaching solution: Compared with the control sample, it showed better growth status and OD value.

[0155] (6) Gel porosity after freeze drying (%): 69

[0156] Example 9 is the same as Example 1, except that the ratios of (I), (II) and (III) are different.

[0157] (1)-(3) are the same as in Example 1.

[0158] (4) Preparation of cold-setting resin calcium glycerophosphate composite gel: (I), (II) and (III) are sterilized with ethylene oxide. 200 ml (I), 150 ml (II) and 200 ml (III) are measured, (I) is added to (II), and (III) is then added to the mixture of (I) and (II), and the mixture is stirred and ultrasonicated for 3 minutes. The temperature is slowly raised to 60°C under nitrogen protection and maintained for 15 minutes, and then transferred to round, rectangular and cube molds and maintained for 5 hours, and then the gel is taken out and sliced ​​or cut.

[0159] (5) Same as Example 1.

[0160] Test results:

[0161] (1) Compressive strength and elasticity (MPa,%): 1.02,38

[0162] (2) Degradation results (%): 1D: 7.7; 1w: 21.3; 2w: 44.4; 3w: 57.6; 4w: 82.1

[0163] (3) pH measurement: 1D: 7.13; 1w: 7.26; 2w: 7.31; 3w: 7.10; 4w: 7.34

[0164] (4) Cytotoxicity and cell proliferation rate (%) test: Level 0; 102 (compared with blank)

[0165] (5) Cultivation and growth status of bone cells in gel leaching solution: Compared with the control sample, it showed better growth status and OD value.

[0166] (6) Gel porosity after freeze drying (%): 75

[0167] Example 10 (same as Example 1, with different ratios of (I), (II) and (III))

[0168] (1)-(3) are the same as in Example 1.

[0169] (4) Preparation of cold-setting resin calcium glycerophosphate composite gel: (I), (II) and (III) are sterilized with ethylene oxide. 200 ml (I), 200 ml (II) and 200 ml (III) are measured, (I) is added to (II), and (III) is then added to the mixture of (I) and (II), and the mixture is stirred and ultrasonicated for 3 minutes. The temperature is slowly raised to 60°C under nitrogen protection and maintained for 15 minutes, and then transferred to round, rectangular and cube molds and maintained for 5 hours, and then the gel is taken out and sliced ​​or cut.

[0170] (5) Same as Example 1.

[0171] Test results:

[0172] (1) Compressive strength and elasticity (MPa,%): 1.14,43

[0173] (2) Degradation results (%): 1D: 8.5; 1w: 22.7; 2w: 44.9; 3w: 58.5; 4w: 82.7

[0174] (3) pH measurement: 1D: 7.10; 1w: 7.25; 2w: 7.32; 3w: 7.34; 4w: 7.34

[0175] (4) Cytotoxicity and cell proliferation rate (%) test: Level 0; 106 (compared with blank)

[0176] (5) Cultivation and growth status of bone cells in gel leaching solution: Compared with the control sample, it showed better growth status and OD value.

[0177] (6) Gel porosity after freeze drying (%): 72

[0178] Comparative Example 1: Cold-setting resin is used as a cancellous bone or cartilage tissue repair material.

[0179] Accurately weigh 10.0g of cold-bonded resin and add it to a 1L volumetric flask. Then add 600ml of water that has been tested for endotoxins. Ultrasonic centrifuge in 20℃ water for 15 minutes to dissolve it completely. Then add water to the 1L mark and ultrasonicate for another 5 minutes. The clear and transparent solution was left overnight. It could not form a gel and could not solidify, making it difficult to proceed to the next step of testing.

[0180] Comparative Example 2: Sodium hyaluronate is used as a cancellous bone or cartilage tissue repair material.

[0181] Accurately weigh 10.0 g of high molecular weight sodium hyaluronate (molecular weight range: 1,800,000-2,200,000) and 5.0 g of medium molecular weight sodium hyaluronate (molecular weight range: 1,000,000-1,800,000) and add them to a 1L volume, then add 600 ml of endotoxin-tested water, ultrasonically centrifuge in 20°C water for 15 minutes to completely dissolve, then add water to the 1L mark, ultrasonically centrifuge for another 5 minutes, and leave the formed clear and transparent solution overnight. It cannot form a gel and cannot solidify, making it difficult to proceed to the next step of testing.

[0182] Comparative Example 3: Same as Example 1, except that sodium hyaluronate was removed.

[0183] Test results:

[0184] (1) Compressive strength and elasticity (MPa,%): 0.41, 35, tensile strength reduced by 50%

[0185] (2) Degradation results (%): 1D: 12.; 1w: 25.5; 2w: 51.9; 3w: 69.4; 4w: 100, degradation is too fast

[0186] (3) pH measurement: 1D: 7.01; 1w: 7.12; 2w: 7.25; 3w: 7.34; 4w: 7.34

[0187] (4) Cytotoxicity and cell proliferation rate (%) test: Level 0; 96 (compared with blank)

[0188] (5) Cultivation and growth status of bone cells in gel leaching solution: Compared with the control sample, it showed better growth status and OD value.

[0189] (6) Gel porosity after freeze drying (%): 80

[0190] Comparative Example 4: Same as Example 1, except that the cold-bonded resin is removed.

[0191] Test results:

[0192] (1) Compressive strength and elasticity (MPa,%): 0.32, 41, tensile strength reduced by 60%

[0193] (2) Degradation results (%): 1D: 134; 1w: 27.3; 2w: 54.4; 3w: 75.2; 4w: 100, degradation is too fast

[0194] (3) pH measurement: 1D: 7.01; 1w: 7.09; 2w: 7.17; 3w: 7.21; 4w: 7.25

[0195] (4) Cytotoxicity and cell proliferation rate (%) test: Level 0; 97 (compared with blank)

[0196] (5) Cultivation and growth status of bone cells in gel leaching solution: Compared with the control sample, it showed better growth status and OD value.

[0197] (6) Gel porosity after freeze drying (%): 81.

[0198] Comparative Example 5: Same as Example 1, except that the cold-gel-removing resin and sodium hyaluronate were removed.

[0199] Unable to form gel and unable to enter gel test.

[0200] Comparative Example 6: Same as Example 1, except that calcium glycerophosphate, calcium saccharate and amino acid calcium are removed.

[0201] After the cold-setting resin solution and the sodium hyaluronate solution were mixed, they could not form a gel and could not enter the gel test.

[0202] It should be noted that the preferred embodiments of the present invention are given in the specification of the present invention, but the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not intended to be additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features continue to be combined with each other to form various embodiments not listed above, which are all considered to be within the scope of the present invention specification; further, for ordinary technicians in this field, they can be improved or transformed according to the above description, and all these improvements and transformations should belong to the protection scope of the claims attached to the present invention.

Claims

1. A bone repair implant based on cold-setting resin calcium glycerophosphate composite gel, characterized in that: The bone repair implant comprises components A, B and C, wherein component A is cold setting resin, component B is sodium hyaluronate, and component C is calcium glycerophosphate, magnesium citrate, calcium saccharate and amino acid calcium, wherein components A, B and C form a composite gel.

2. The bone repair implant according to claim 1, characterized in that: The sodium hyaluronate includes high molecular weight sodium hyaluronate and medium molecular weight sodium hyaluronate; Further, the molecular weight range of the high molecular weight sodium hyaluronate is 1800000 to 2200000, and the molecular weight range of the medium molecular weight sodium hyaluronate is 1000000 to 1800 000; Furthermore, the mass ratio of the high molecular weight sodium hyaluronate to the medium molecular weight sodium hyaluronate is 0.5-2:2-0.

5.

3. The bone repair implant according to claim 1, characterized in that: Glycerophosphate calcium includes one or more of β-glycerophosphate calcium, D(+)α-glycerophosphate calcium and L(-)α-glycerophosphate calcium; Further, the calcium saccharate includes one or both of calcium gluconate and calcium L-threonate; Furthermore, the amino acid calcium includes one or more of calcium glycinate, calcium lysine, calcium L-aspartate, calcium aspartate, calcium glutamate and calcium L-histidine.

4. A method for preparing a bone repair implant based on a cold-setting resin calcium glycerophosphate composite gel according to any one of claims 1 to 3, characterized in that: The method comprises preparing aqueous solutions of components A, B and C respectively, first mixing the aqueous solutions of components A and B, then adding the aqueous solution of component C, stirring and mixing, and then standing to form a gel, and then freeze-drying to obtain the bone repair implant based on the cold-setting resin calcium glycerophosphate composite gel.

5. The preparation method according to claim 4, characterized in that: The aqueous solutions of component A, component B and component C are mixed in a volume ratio of 1.0-2.0:1.0-2.0:0.5-2.

5.

6. The preparation method according to claim 4, characterized in that: The aqueous solution of component A is prepared as follows: dissolve the cold-bonding resin in water that has passed the endotoxin test at a concentration of 1.0-20 g / L to obtain a clear and transparent solution for use; Furthermore, the dissolving is performed at 0-80° C. under ultrasound or stirring.

7. The preparation method according to claim 4, characterized in that: The aqueous solution of component B is prepared as follows: sodium hyaluronate is dissolved in water that has passed the endotoxin test at a concentration of 5.0-20.0 g / L to obtain a clear and transparent solution for use; Furthermore, the dissolving is performed at 0-80° C. under ultrasound or stirring.

8. The preparation method according to claim 4, characterized in that: The aqueous solution of component C is prepared as follows: calcium glycerophosphate is dissolved in water that has passed endotoxin testing at a concentration of 10.0-25.0 g / L to obtain a clear and transparent solution I; magnesium citrate, calcium saccharate and amino acid calcium are dissolved in water that has passed endotoxin testing at a concentration of 5.0-25.0 g / 100 ml to obtain a clear and transparent solution II; solution I and solution II are mixed at a volume ratio of 1.0:0.5-2.5 to obtain an aqueous solution of component C and stored at below 10° C. for use; Further, the dissolution of calcium glycerophosphate is carried out at below 20° C. under ultrasound or stirring; Further, the mass ratio of magnesium citrate, calcium saccharate and amino acid calcium is 10-25:20-50:70-150; Furthermore, the dissolution of magnesium citrate, calcium saccharate and calcium amino acid is carried out at room temperature under ultrasound or stirring.

9. The preparation method according to claim 4, characterized in that: The step of standing to form a gel comprises first heating the mixed aqueous solution of components A, B and C to 50-80° C. under nitrogen protection, maintaining the mixture for 15-45 minutes, then transferring the mixture to a mold, cooling the mixture to room temperature, and maintaining the mixture for 2-10 hours; Furthermore, the preparation method further comprises sterilizing the aqueous solutions of components A, B and C before mixing them, such as ethylene oxide sterilization; Furthermore, the preparation method also includes slicing or cutting the formed gel before freeze-drying to obtain a desired shape and size.

10. Use of the bone repair implant based on cold-setting resin calcium glycerophosphate composite gel according to any one of claims 1 to 3 or the bone repair implant based on cold-setting resin calcium glycerophosphate composite gel prepared by the preparation method according to any one of claims 4 to 9 in cancellous bone and / or cartilage repair.

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