A bone repair implant based on cold-setting resin calcium glycerophosphate composite gel and its preparation method

Abstract

This invention belongs to the field of bone repair materials technology, specifically relating to a bone repair implant based on a cold-cured resin-calcium glycerophosphate composite gel and its preparation method. The bone repair implant of this invention uses cold-cured resin as the main component of the gel, hyaluronic acid as an auxiliary component, and calcium glycerophosphate, citrate, calcium gluconate, and calcium amino acid as gel solidification promoters to form a bone repair implant with high elasticity, high toughness, and a certain strength. It has been proven that this implant can be used as a basic material for cartilage tissue repair, providing an effective option for the repair of articular cartilage and other cartilage tissues.

Description

Technical Field

[0001] This invention belongs to the field of bone repair materials technology, specifically relating to a bone repair implant based on cold-cured resin glycerol phosphate calcium composite gel and its preparation method. Background Technology

[0002] The human knee joint has a complex structure and function, and bears heavy loads daily, making it susceptible to various acute injuries and chronic strain. One problem with knee injuries is that, compared to bone, damage to articular cartilage is much more difficult to heal. After immobilization, fractured bones can usually heal gradually, but the regenerative capacity of joint cartilage tissue is very poor. After wear and tear, it's difficult to expect it to return to its original state through its own repair mechanisms. The main reasons are: 1. Lack of blood vessels prevents undifferentiated cells from entering the injured site to undergo a healing process similar to other tissues; 2. Cartilage tissue itself lacks undifferentiated cells for repair; 3. Chondrocytes are embedded in a dense collagenous polysaccharide matrix, limiting their own cell proliferation and migration capabilities; 4. Although the synthetic capacity of mature chondrocyte matrix increases when cartilage is damaged, its function is limited and insufficient to meet the needs of repair.

[0003] Treatment methods for articular cartilage injury mainly include: 1. General treatment. General treatment mainly includes muscle function exercises to stabilize the joint, joint braces or braces to protect the joint, reducing the range of motion of the joint, and intra-articular injection of lubricant. 2. Drug treatment. Drug treatment mainly selects non-steroidal anti-inflammatory drugs and drugs with liver and kidney tonifying and blood-activating effects. Drugs with liver and kidney tonifying and blood-activating effects have a certain auxiliary effect on cartilage repair. 3. Surgical treatment. Surgical treatment mainly includes localized cartilage resection with drilling, bone realignment surgery to correct the alignment, artificial joint replacement, cartilage grafting, and bone resection. 4. Other treatments. Other treatments mainly include paraffin wax therapy and shortwave diathermy.

[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 been a challenge for patients and clinicians. Currently, many researchers are exploring methods to repair damaged articular cartilage, such as using stem cells to promote tissue repair; however, these efforts have not yet been entirely successful. At present, protecting the knee and preventing injury remains the best solution. Controlling weight, engaging in appropriate physical activity, and strengthening lower limb muscles can all help reduce wear and tear on the knee cartilage.

[0006] Since Chestman and Smith first began researching the in vitro culture of chondrocytes for cartilage defect repair in 1965, a new understanding has emerged regarding the concept that articular cartilage damage cannot be repaired through its own cartilage proliferation. Experiments have shown that not only young but also old articular cartilage specimens can still produce new hyaline cartilage in vitro. Although the proliferative capacity of chondrocytes is limited, their quality and quantity directly affect the in vitro culture expansion effect, and the biological characteristics exhibited by chondrocytes vary depending on the growth environment. Chondrocytes grow well and maintain phenotypic stability in suspension culture or semi-solid agar medium. When cultured for a long time in a four-dimensional culture environment with hydrogel covering the bottom of the culture flask, chondrocytes can form nodular structures, and their cell morphology, extracellular matrix secretion, and cartilage-specific gene expression are similar to those of articular cartilage.

[0007] In recent years, with the deepening of research on bone repair materials, the focus has gradually expanded from bone tissue regeneration and repair to cartilage tissue repair, attempting to use biomaterials to repair damaged cartilage tissue and restore its function. These attempts include hyaluronic acid injection into joints for lubrication, treatment with glucosamine and chondroitin sulfate, and hybrid therapy combining stem cells and materials. Hundreds of thousands of papers have been published on these attempts, but materials suitable for clinical treatment and cartilage repair are extremely rare. This indicates that the most critical problem hindering cartilage repair—the discovery of key foundational materials—remains unsolved, and the mechanism of synergistic action between materials and stem cells is not fully understood. Therefore, it is necessary to find more effective materials as the foundation for cartilage repair.

[0008] Gel gum is a polymer of glucuronic acid, 6-deoxy-L-mannopyranoside, and D-glucuronide, in the form of calcium, potassium, and sodium salts. Also known as gellan gum, CAS number 71010-52-1, it is colorless, transparent, and highly resilient. It is a major component in the preparation of plant tissue culture media and microbial culture media. It is a substitute for agar secreted by Pseudomonas bacteria and is characterized by its colorlessness, transparency, and high toughness.

[0009] Sodium hyaluronate is a physiologically active substance widely found in the human body. It is a high-molecular-weight linear mucopolysaccharide polymerized from disaccharide units composed of glucuronic acid and acetaminohexose, with a molecular weight of 1 million. In water, it forms a viscous, elastic solution with physiological pH and ionic strength. Sodium hyaluronate is a major component of synovial fluid and a component of cartilage matrix. Within the joint cavity, it acts as a lubricant, reducing friction between tissues. Simultaneously, it exerts its elastic effect, buffering stress on articular cartilage and fulfilling its physiological functions.

[0010] Calcium glycerophosphate is a white crystalline powder. It is odorless, almost tasteless, slightly bitter, and hygroscopic. It plays a role in the formation of cellular structures and bone tissue, and also contributes to the metabolism of calcium glycerophosphate salts, thus boosting brain function.

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

[0012] Based on the above background, this invention designs a bone repair implant based on a composite gel. Plant-based gel, namely cold-curing resin, is selected as the main component of the gel, sodium hyaluronate is selected as the auxiliary component, and calcium glycerophosphate, citrate, calcium gluconate, and calcium amino acid are used as gel solidification promoters to form a bone repair implant with high elasticity, high toughness, and certain strength. This is intended as a basic material for cartilage tissue repair, providing an effective option 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-cured resin-calcium glycerophosphate composite gel, comprising components A, B and C, wherein component A is a cold-cured resin, component B is sodium hyaluronate, and component C is calcium glycerophosphate, magnesium citrate, calcium gluconate and calcium amino acid, 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, calcium glycerophosphate includes one or more of β-calcium glycerophosphate, D(+)α-calcium glycerophosphate, and L(-)α-calcium glycerophosphate.

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

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

[0020] In another aspect, the present invention provides a method for preparing a bone repair implant based on a cold-cured resin glycerophosphate calcium composite gel as described herein, comprising 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 thoroughly, allowing it to stand to form a gel, and then freeze-drying to obtain the bone repair implant based on the cold-cured resin glycerophosphate calcium composite gel.

[0021] Furthermore, aqueous solutions of components A, B, and 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: the cold-curing resin is dissolved in water that has passed endotoxin detection at a concentration of 1.0-20 g / L to obtain a clear and transparent solution for later use; further, the dissolution is carried out at 0-80°C under ultrasonic or stirring conditions.

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

[0024] Further, 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 gluconate, and calcium amino acids 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; solutions I and II are mixed in a volume ratio of 1.0:0.5-2.5 to obtain an aqueous solution of component C, which is then stored below 10°C for later use.

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

[0026] Furthermore, the dissolution of calcium glycerophosphate is carried out at below 20°C under ultrasonic or stirring conditions.

[0027] Furthermore, the dissolution of magnesium citrate, calcium gluconate, and calcium amino acid was carried out at room temperature under ultrasonic or stirring conditions.

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

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

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

[0031] Beneficial effects of the invention

[0032] This invention designs a bone repair implant based on a cold-cured resin-calcium glycerophosphate composite gel. Cold-cured resin is selected as the main component of the gel, hyaluronic acid is selected as the auxiliary component, and calcium glycerophosphate, citrate, calcium gluconate and calcium amino acid are used as gel solidification promoters to form a bone repair implant with high elasticity, high toughness and certain strength. It has been proven that it can be used as a basic material for cartilage tissue repair, providing an effective option for the repair of articular cartilage and other cartilage tissues. Detailed Implementation

[0033] This invention relates to a bone repair implant based on a cold-cured resin-calcium glycerophosphate composite gel and its preparation method, particularly related to the repair and functional recovery of cancellous bone and cartilage tissues. Based on the composition, structure, and performance characteristics of cancellous bone and cartilage tissues, cold-cured 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 gluconate, and calcium amino acid are used as gel solidification accelerators to form a bone repair implant with high elasticity, high toughness, and a certain strength.

[0034] To obtain a bone repair implant based on a cold-cured resin glycerol phosphate calcium composite gel, the present invention provides the following specific implementation technical solution.

[0035] First, cold-curing resin is selected as the main component of the gel. A cold-curing resin solution (I) is prepared using tested water (the water used in this 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, endotoxin can be determined by colorimetry) with a concentration of 1.0-20 g / L, preferably 1.5-10 g / L.

[0036] Specifically, use cold-curing resin with a purity of ≥99%. Accurately weigh 1.0-20g of cold-curing resin (accurate to 0.01g) and add it to a 1L volumetric flask. Then add 600ml of water that has been tested for endotoxins. Sonicate in water at 0-80℃ for 10-30 minutes or stir until completely dissolved. Then add water to the 1L mark and sonicate for another 3-5 minutes or stir. Set aside the resulting clear and transparent solution.

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

[0038]

[0039] Sodium hyaluronate is a high-molecular-weight saccharide compound composed of repeating units of varying molecular weights. It is a white or off-white granule or powder, odorless, and when dry, contains 2.8%-4.0% nitrogen and 37.0%-51.0% glucuronic acid. Hyaluronic acid includes high molecular weight hyaluronic acid (molecular weight range 1,800,000-2,200,000); medium molecular weight hyaluronic acid (molecular weight range 1,000,000-1,800,000); and low molecular weight hyaluronic acid (molecular weight range 400,000-1,000,000). The molecular weight significantly affects its viscosity, moisturizing properties, etc. Therefore, different molecular weight sodium hyaluronates result in gels with vastly different properties. Since the repair of cartilage and cancellous bone has a long cycle, the gel needs to maintain its shape for a relatively long time. Therefore, this invention selects high molecular weight and medium molecular weight sodium hyaluronate as components of the gel.

[0040] Prepare an aqueous solution (II) of sodium hyaluronate, consisting of high molecular weight sodium hyaluronate and medium molecular weight sodium hyaluronate, using tested water. The concentration of this solution is 5.0-20.0 g / L, preferably 10-15 g / L. 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 cleanroom, accurately weigh 10g of high molecular weight sodium hyaluronate and 5g of medium molecular weight sodium hyaluronate. Add the weighed high molecular weight and medium molecular weight sodium hyaluronate to a 1L volume, then add 600ml of water that has undergone endotoxin testing. Sonicate in water at 0-80℃ for 10-30 minutes or stir until completely dissolved. Then add water to the 1L mark, and sonicate again for 3-5 minutes or stir. The resulting clear and transparent solution is ready for use.

[0042] Furthermore, the present invention uses calcium glycerophosphate. As a gel formation promoter, calcium glycerophosphate exists in three isomers: β-calcium glycerophosphate ((HOCH2)2CHOPO3Ca), D(+), and L(-)α-calcium glycerophosphate (HOCH2CH(OH)CH2OPO3Ca). One or more of these isomers can be used simultaneously. This invention uses it as a calcium ion donor to accelerate the gelation process, and also as a calcium and phosphorus donor for bone tissue formation. This invention prepares it as an aqueous solution with a concentration of 10.0-25.0 g / L. Specifically, in a cleanroom, accurately weigh 10.0-25.0 g of calcium glycerophosphate and add it to a 1 L volumetric flask. Then add 600 ml of water (tested for endotoxins) and sonicate at below 20°C for 10-30 minutes or stir until completely dissolved. Then add water to the 1 L mark, and sonicate again for 5-30 minutes or stir. The resulting clear, transparent solution is then ready for use. The aqueous solution of calcium glycerophosphate should be prepared below 20°C and stored at no higher than 10°C.

[0043] Magnesium citrate was also selected. Calcium gluconate L-Threonate Calcium Calcium and magnesium salts with high solubility, such as calcium amino acid salts, are used as gel formation promoters and calcium-phosphorus ratio regulators. Preferred calcium amino acid salts are those that are soluble in water and have high water solubility, such as calcium glycine. Lysine Calcium L-aspartate calcium Calcium aspartate Calcium glutamate L-Histidine Calcium And, but not limited to, the above-mentioned amino acid calcium.

[0044] This invention prepares it as an aqueous solution with a concentration of 5.0-25.0 g / 100 ml. Specifically, in a cleanroom, accurately weigh 50.0-250.0 g of one or more of magnesium citrate, calcium gluconate, L-threonate calcium, and amino acid calcium, add them to a 1 L volumetric flask, then add 600 ml of water that has undergone endotoxin testing, sonicate in room temperature water for 5-30 minutes until completely dissolved, then add water to the 1 L mark, sonicate again for 5-30 minutes, and use the resulting clear and transparent solution.

[0045] Furthermore, aqueous solutions of magnesium citrate, calcium gluconate, and calcium amino acid were mixed with an aqueous solution of calcium glycerophosphate to form a composite aqueous solution (III) enriched with calcium, phosphorus, sugars, and amino acids. Considering the co-solubility of various anions and cations after mixing and the effective content of calcium, phosphorus, sugars, and amino acids in the solution, the ratio of the aqueous solution of magnesium citrate, calcium gluconate, and calcium amino acid to the aqueous solution of calcium glycerophosphate was (V) 0.5-2.5:1.0. Within this ratio range, a high concentration of coexisting anions and cations can be formed without precipitation, providing a favorable chemical and physical environment for gel preparation.

[0046] Further, the composite solution (III) formed by the cold-curing resin solution (I), sodium hyaluronate solution (II), and magnesium citrate, calcium gluconate, and calcium amino acid solution with glycerophosphate solution is sterilized with ethylene oxide. (I), (II), and (III) are mixed in a volume ratio (V) of 1.0-2.0:1.0-2.0:0.5-2.5. Specifically, (I) is added to (II) while stirring, and then (III) is added to the mixture of (I) and (II). After stirring for 5-10 minutes, the mixture is allowed to stand to form a gel. The temperature of (I), (II), and (III) before addition is preferably no higher than 10°C. After mixing, the temperature is slowly raised to 60°C under nitrogen protection and maintained for 15-45 minutes. The mixture is then transferred to circular, cuboid, and cubic molds, cooled to room temperature, and maintained for 2-10 hours. The gel is then removed and sliced ​​or cut to obtain the desired shape and size.

[0047] Furthermore, partial slices or cut gels are freeze-dried to obtain bone repair implants based on porous cryo-cured resin glycerol phosphate composite gels. Traditional drying causes gel shrinkage, wrinkling, and damage to the gel structure, while the structure of the sample is not destroyed during freeze-drying because the solid components are supported by the ice in their place. When the ice sublimates, pores are left in the dried residue. This preserves the integrity of the product's biological and chemical structure and its activity. Therefore, this invention uses freeze-drying to dry the formed cryo-cured resin glycerol phosphate composite gel bone repair implants, forming a porous gel material that provides space for blood vessel ingrowth and enhances the physiological activity of the cryo-cured resin glycerol phosphate composite gel bone repair implants.

[0048] Furthermore, the performance of the obtained cold-cured resin glycerol calcium phosphate composite gel was tested:

[0049] (1) Compression test method: Loading speed 1mm / min, compression strength: The mechanical strength of the sample (diameter 6mm, height 12mm) is measured by a universal mechanical testing instrument. Each sample is measured at least 5 times.

[0050] (2) Degradation test method: The injection-molded material was subjected to degradation test in PBS solution. The PBS preparation method is as follows: accurately weigh KH2PO4 (0.544g), Na2HPO4·12H2O (7.16g), NaCl (16g) and KCl (0.402g) and dissolve them in 2L of deionized water, and make up to volume with a volumetric flask. The pH measurement range is 7.2-7.4. Place the sample in 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 shaking incubator at 37℃ and 80rpm / min. Take out the sample at 1 (1D), 1w, 2w, 3w and 4w, weigh it and calculate the weight loss rate. Three control groups were set up for each sample.

[0051] (3) pH measurement: pH meter was used to measure the supernatant of the sample. PBS was replaced every 7 days.

[0052] (4) Cytotoxicity and cell proliferation rate test: The extract was prepared in accordance with the provisions of T16886 concerning biological materials, and the proliferation rate was calculated by comparing the standard extract with the blank.

[0053] (5) Culture and growth of osteocytes in gel extract.

[0054] The prepared cold-cured resin glycerol phosphate composite gel was immersed in Dulbecco modified Eagle medium (DMEM), and a 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 added at a concentration of 1×10⁻⁶. 3 rBMSCs were seeded at a density of 5 × 10⁶ cells / well in 96-well plates. Cell viability was quantitatively determined by CCK-8 assay on days 1, 3, and 5, and absorbance was read at 450 nm using a microplate reader. Cell morphology was observed using an inverted fluorescence microscope after staining with rhodamine-labeled phalloidin (FITC) and 4,6-diamidinyl-2-phenylindole (DAPI). Alkaline phosphatase (ALP) activity was used to detect early osteogenic differentiation. rBMSCs were seeded at a density of 5 × 10⁶ cells / well. 4 Cells were seeded at a density of 10 cells / well in 24-well plates and cultured in extract supplemented with osteogenic differentiation medium (0.05 mmol / L vitamin C, 10 mmol / L β-glycerophosphate, and 0.1 μmol / L dexamethasone). Cells were cultured at 7 and 11 days according to the ALP kit protocol. Final ALP activity was normalized to protein concentration using the BCA kit. Alizarin Red staining was used to determine cell mineralization in late differentiation. rBMSCs were seeded at 5 × 10⁻⁶ cells / well. 4Cells were seeded at a density of 10 cells / well in 24-well plates. After 7 and 14 days, cells were washed with PBS, fixed with 4% paraformaldehyde for 20 minutes, and stained with alizarin red for 20 minutes at room temperature. The stained nodules were incubated in 10% hexadecylpyridine chloride for 30 minutes and measured at 620 nm using a microplate reader.

[0055] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0056] Example 1

[0057] (1) Preparation of cold-curing resin solution: Accurately weigh 10.0g of cold-curing resin and add it to a 1L volumetric flask. Then add 600ml of water that has been tested for endotoxins. Sonicate in water at 20℃ for 15 minutes to dissolve completely. Then add water to the 1L mark and sonicate for another 5 minutes. The resulting clear and transparent solution is ready for use.

[0058] (2) Sodium hyaluronate solution (II): Accurately weigh 10.0g of high molecular weight sodium hyaluronate (molecular weight range 1,800,000 to 2,200,000) and 5.0g of medium molecular weight sodium hyaluronate (molecular weight range 1,000,000 to 1,800,000) and add them to a 1L volume. Then add 600ml of water that has been tested for endotoxins. Centrifuge in water at 20°C for 15 minutes to dissolve completely. Then add water to the 1L mark and sonicate for another 5 minutes. The resulting clear and transparent solution is ready for use.

[0059] (3) Composite solution (III) formed by magnesium citrate, calcium gluconate, and calcium amino acid solution with calcium glycerophosphate solution: Accurately weigh 15g of β-calcium glycerophosphate and add it to a 1L volumetric flask. Then add 600ml of water that has been tested for endotoxins. Sonicate in water at 20℃ for 15 minutes until completely dissolved. Then add water to the 1L mark and sonicate for another 10 minutes. The resulting clear and transparent solution is ready for use. Store below 10℃ for later use.

[0060] Accurately weigh 10.0g magnesium citrate, 20.0g calcium gluconate, 30.0g calcium glycine, 30.0g calcium lysine, and 10.0g calcium phenylalanine, and add them to a 1L volumetric flask. Then add 600ml of water that has been tested for endotoxins. Sonicate the solution in room temperature water for 10 minutes until completely dissolved. Then add water to the 1L mark and sonicate for another 10 minutes. Set aside the clear and transparent solution.

[0061] Take 400 ml of β-glycerophosphate calcium aqueous solution and 200 ml of magnesium citrate, calcium gluconate and amino acid calcium aqueous solution, mix them and sonicate for 5 minutes to form a complex aqueous solution of magnesium citrate, calcium gluconate and amino acid calcium and glycerophosphate calcium (III).

[0062] (4) Preparation of cold-cured resin glycerol phosphate composite gel: (I), (II) and (III) were sterilized with ethylene oxide. Measure 150 ml (I), 150 ml (II) and 200 ml (III), add (I) to (II), and then add (III) to the mixture of (I) and (II), stir and sonicate for 3 minutes, slowly raise the temperature to 60°C under nitrogen protection and maintain for 15 minutes, then transfer to circular, cuboid and cubic molds, maintain for 5 hours, and then take out the gel for slicing or cutting.

[0063] (5) Preparation of porous cold-cured resin glycerol phosphate calcium composite gel: The slices or cut gels from part (4) were freeze-dried to obtain porous cold-cured resin glycerol phosphate calcium composite gel bone repair implant.

[0064] (6) Performance test of cold-cured resin glycerol phosphate calcium composite gel. The test contents of the above implementation plan were carried out as follows: (1) compression test method; (2) degradation test; (3) pH measurement; (4) cytotoxicity and cell proliferation rate test; (5) culture and growth status of osteocytes 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: 1 day: 7.05; 1 week: 7.21; 2 weeks: 7.25; 3 weeks: 7.10; 4 weeks: 7.35

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

[0070] (5) Culture and growth status of osteoblasts in gel extract: Compared with the control, they showed better growth status and OD value.

[0071] (6) Porosity of the freeze-dried gel (%): 78

[0072] Example 2 (same as Example 1, but with a 50% increase in the concentration of the cold-curing resin aqueous solution)

[0073] (1) Preparation of cold-curing resin solution: Accurately weigh 15.0g of cold-curing resin and add it to a 1L volumetric flask. Then add 600ml of water that has been tested for endotoxins. Sonicate in water at 20℃ for 15 minutes to dissolve completely. Then add water to the 1L mark and sonicate for another 5 minutes. The resulting clear and transparent solution is ready for use.

[0074] (2)-(5) Same as 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: 1 day: 7.02; 1 week: 7.23; 2 weeks: 7.27; 3 weeks: 7.22; 4 weeks: 7.33

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

[0080] (5) Culture and growth status of osteoblasts in gel extract: Compared with the control, they showed better growth status and OD value.

[0081] (6) Porosity of the freeze-dried gel (%): 70

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

[0083] (1) Same as Example 1

[0084] (2) Sodium hyaluronate solution (II): Accurately weigh 15.0g of high molecular weight sodium hyaluronate (molecular weight range 1,800,000~2,200,000) and 7.50g of medium molecular weight sodium hyaluronate (molecular weight range 1,000,000~1,800,000) and add them to a 1L volume. Then add 600ml of water that has been tested for endotoxins. Centrifuge in water at 20℃ for 15 minutes to dissolve completely. Then add water to the 1L mark and sonicate for another 5 minutes. The resulting clear and transparent solution is ready for 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: 1 day: 7.01; 1 week: 7.23; 2 weeks: 7.21; 3 weeks: 7.32; 4 weeks: 7.29

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

[0091] (5) Culture and growth status of osteoblasts in gel extract: Compared with the control, they showed better growth status and OD value.

[0092] (6) Porosity of the freeze-dried gel (%): 76

[0093] Example 4 (same as Example 1, except the ratio of high molecular weight / 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 10.0g of high molecular weight sodium hyaluronate (molecular weight range 1,800,000 to 2,200,000) and 10.0g of medium molecular weight sodium hyaluronate (molecular weight range 1,000,000 to 1,800,000) and add them to a 1L volume. Then add 600ml of water that has been tested for endotoxins. Sonicate in water at 20°C for 15 minutes until completely dissolved. Then add water to the 1L mark and sonicate for another 5 minutes. The resulting clear and transparent solution is ready for use.

[0096] (3)-(5) Same as 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: Grade 0; 101 (compared with blank)

[0102] (5) Culture and growth status of osteoblasts in gel extract: Compared with the control, they showed better growth status and OD value.

[0103] (6) Porosity of the freeze-dried gel (%): 65

[0104] Example 5 (same as Example 1, but with a different concentration of glycerol calcium phosphate solution)

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

[0106] (3) Composite solution (III) formed by magnesium citrate, calcium gluconate, and amino acid calcium solution with glycerophosphate calcium solution: Accurately weigh 20.0 g of β-glycerophosphate calcium and add it to a 1 L volumetric flask. Then add 600 ml of water that has been tested for endotoxins. Sonicate in water at 20 °C for 15 minutes until completely dissolved. Then add water to the 1 L mark and sonicate for another 10 minutes. The resulting clear and transparent solution is ready for use. Store below 10 °C for later use.

[0107] Accurately weigh 10.0g magnesium citrate, 20.0g calcium gluconate, 30.0g calcium glycine, 30.0g calcium lysine, and 10.0g calcium phenylalanine, and add them to a 1L volumetric flask. Then add 600ml of water that has been tested for endotoxins. Sonicate the solution in room temperature water for 10 minutes until completely dissolved. Then add water to the 1L mark and sonicate for another 10 minutes. Set aside the clear and transparent solution.

[0108] Take 400 ml of β-glycerophosphate calcium aqueous solution and 200 ml of magnesium citrate, calcium gluconate and amino acid calcium aqueous solution, mix them and sonicate for 5 minutes to form a complex aqueous solution of magnesium citrate, calcium gluconate and amino acid calcium 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: 1 day: 7.05; 1 week: 7.23; 2 weeks: 7.31; 3 weeks: 7.33; 4 weeks: 7.35

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

[0115] (5) Culture and growth status of osteoblasts in gel extract: Compared with the control, they showed better growth status and OD value.

[0116] (6) Porosity of the freeze-dried gel (%): 68

[0117] Example 6 (same as Example 1, except that the concentrations of magnesium citrate, calcium gluconate, and calcium amino acid solutions are different, and increased by 20% proportionally)

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

[0119] (3) Composite solution (III) formed by magnesium citrate, calcium gluconate, and calcium amino acid solution with calcium glycerophosphate solution: Accurately weigh 15g of β-calcium glycerophosphate and add it to a 1L volumetric flask. Then add 600ml of water that has been tested for endotoxins. Sonicate in water at 20℃ for 15 minutes until completely dissolved. Then add water to the 1L mark and sonicate for another 10 minutes. The resulting clear and transparent solution is ready for use. Store below 10℃ for later use.

[0120] Accurately weigh 12.0g magnesium citrate, 24.0g calcium gluconate, 36.0g calcium glycine, 36.0g calcium lysine, and 12.0g calcium phenylalanine, and add them to a 1L volumetric flask. Then add 600ml of water that has been tested for endotoxins. Sonicate the solution in room temperature water for 10 minutes until completely dissolved. Then add water to the 1L mark and sonicate for another 10 minutes. Set aside the clear and transparent solution.

[0121] Take 400 ml of β-glycerophosphate calcium aqueous solution and 200 ml of magnesium citrate, calcium gluconate and amino acid calcium aqueous solution, mix them and sonicate for 5 minutes to form a complex aqueous solution of magnesium citrate, calcium gluconate and amino acid calcium 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: 1 day: 7.12; 1 week: 7.23; 2 weeks: 7.30; 3 weeks: 7.33; 4 weeks: 7.35

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

[0128] (5) Culture and growth status of osteoblasts in gel extract: Compared with the control, they showed better growth status and OD value.

[0129] (6) Porosity of the freeze-dried gel (%): 71

[0130] Example 7 (same as Example 1, except that the ratio of magnesium citrate, calcium gluconate, and calcium amino acid solution to calcium glycerophosphate 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 gluconate, and calcium amino acid solution with calcium glycerophosphate solution: Accurately weigh 15g of β-calcium glycerophosphate and add it to a 1L volumetric flask. Then add 600ml of water that has been tested for endotoxins. Sonicate in water at 20℃ for 15 minutes until completely dissolved. Then add water to the 1L mark and sonicate for another 10 minutes. The resulting clear and transparent solution is ready for use. Store below 10℃ for later use.

[0133] Accurately weigh 10.0g magnesium citrate, 20.0g calcium gluconate, 30.0g calcium glycine, 30.0g calcium lysine, and 10.0g calcium phenylalanine, and add them to a 1L volumetric flask. Then add 600ml of water that has been tested for endotoxins. Sonicate the solution in room temperature water for 10 minutes until completely dissolved. Then add water to the 1L mark and sonicate for another 10 minutes. Set aside the clear and transparent solution.

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

[0135] (4)-(5) Same as 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: 1 day: 7.15; 1 week: 7.29; 2 weeks: 7.32; 3 weeks: 7.32; 4 weeks: 7.36

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

[0141] (5) Culture and growth status of osteoblasts in gel extract: Compared with the control, they showed better growth status and OD value.

[0142] (6) Porosity of the freeze-dried gel (%): 69

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

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

[0145] (3) Composite solution (III) formed by magnesium citrate, L-threonate calcium, and amino acid calcium solution with glycerophosphate calcium solution: Accurately weigh 15g of β-glycerophosphate calcium and add it to a 1L volumetric flask. Then add 600ml of water that has been tested for endotoxins. Sonicate in water at 20℃ for 15 minutes until completely dissolved. Then add water to the 1L mark and sonicate for another 10 minutes. The resulting clear and transparent solution is ready for use. Store below 10℃ for later use.

[0146] Accurately weigh 10.0g magnesium citrate, 20.0g L-threonate calcium, 30.0g calcium glycine, 30.0g L-histidine calcium, and 10.0g calcium phenylalanine, and add them to a 1L volumetric flask. Then add 600ml of water that has been tested for endotoxins. Sonicate the solution in room temperature water for 10 minutes until completely dissolved. Then add water to the 1L mark and sonicate for another 10 minutes. Set aside the clear and transparent solution.

[0147] Take 400 ml of β-glycerophosphate calcium aqueous solution and 200 ml of magnesium citrate, calcium gluconate and amino acid calcium aqueous solution, mix them and sonicate for 5 minutes to form a complex aqueous solution of magnesium citrate, calcium gluconate and amino acid calcium 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: 1 day: 7.11; 1 week: 7.25; 2 weeks: 7.32; 3 weeks: 7.33; 4 weeks: 7.34

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

[0154] (5) Culture and growth status of osteoblasts in gel extract: Compared with the control, they showed better growth status and OD value.

[0155] (6) Porosity of the freeze-dried gel (%): 69

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

[0157] (1)-(3) Same as Example 1.

[0158] (4) Preparation of cold-cured resin glycerol phosphate composite gel: (I), (II) and (III) were sterilized with ethylene oxide. Measure 200 ml (I), 150 ml (II) and 200 ml (III), add (I) to (II), and then add (III) to the mixture of (I) and (II), stir and sonicate for 3 minutes, slowly raise the temperature to 60°C under nitrogen protection and maintain for 15 minutes, then transfer to circular, cuboid and cubic molds, maintain for 5 hours, and then take out the gel for slicing or cutting.

[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: 1 day: 7.13; 1 week: 7.26; 2 weeks: 7.31; 3 weeks: 7.10; 4 weeks: 7.34

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

[0165] (5) Culture and growth status of osteoblasts in gel extract: Compared with the control, they showed better growth status and OD value.

[0166] (6) Porosity of the freeze-dried gel (%): 75

[0167] Example 10 (same as Example 1, except that the proportions of (I), (II) and (III) are different)

[0168] (1)-(3) Same as Example 1.

[0169] (4) Preparation of cold-cured resin glycerol phosphate composite gel: (I), (II) and (III) were sterilized with ethylene oxide. Measure 200 ml of (I), 200 ml of (II) and 200 ml of (III). Add (I) to (II), and then add (III) to the mixture of (I) and (II). Stir and sonicate for 3 minutes. Under nitrogen protection, slowly raise the temperature to 60°C and maintain for 15 minutes. Then transfer to circular, cuboid and cubic molds and maintain for 5 hours. Then take out the gel and slice or cut it.

[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: 1 day: 7.10; 1 week: 7.25; 2 weeks: 7.32; 3 weeks: 7.34; 4 weeks: 7.34

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

[0176] (5) Culture and growth status of osteoblasts in gel extract: Compared with the control, they showed better growth status and OD value.

[0177] (6) Porosity of the freeze-dried gel (%): 72

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

[0179] Accurately weigh 10.0g of cold-curing resin and add it to a 1L volumetric flask. Then add 600ml of water that has been tested for endotoxins. Sonicate the solution in 20℃ water for 15 minutes until it is completely dissolved. Then add water to the 1L mark and sonicate for another 5 minutes. Let the resulting clear and transparent solution stand overnight. If it cannot form a gel or solidify, it is difficult to proceed to the next step of the test.

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

[0181] Accurately weigh 10.0g of high molecular weight sodium hyaluronate (molecular weight range 1,800,000–2,200,000) and 5.0g of medium molecular weight sodium hyaluronate (molecular weight range 1,000,000–1,800,000) and add them to a 1L volume. Then add 600ml of water that has been tested for endotoxins. Sonicate the solution in water at 20°C for 15 minutes until completely dissolved. Then add water to the 1L mark and sonicate for another 5 minutes. Let the resulting clear and transparent solution stand overnight. If it cannot form a gel or solidify, it is difficult to proceed to the next step of the test.

[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 decreased by 50%.

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

[0186] (3) pH measurement: 1 day: 7.01; 1 week: 7.12; 2 weeks: 7.25; 3 weeks: 7.34; 4 weeks: 7.34

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

[0188] (5) Culture and growth status of osteoblasts in gel extract: Compared with the control, they showed better growth status and OD value.

[0189] (6) Porosity of the freeze-dried gel (%): 80

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

[0191] Test results:

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

[0193] (2) Degradation results (%): 1D: 134; 1w: 27.3; 2w: 54.4; 3w: 75.2; 4w: 100, degradation was 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: Grade 0; 97 (compared with blank)

[0196] (5) Culture and growth status of osteoblasts in gel extract: Compared with the control, they showed better growth status and OD value.

[0197] (6) Porosity of the freeze-dried gel (%): 81.

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

[0199] Unable to form a gel, thus unable to proceed to the gel test.

[0200] Comparative Example 6: Same as Example 1, except that calcium glycerophosphate, calcium gluconate, and calcium amino acids were removed.

[0201] When cold-curing resin solution and sodium hyaluronate solution are mixed, they cannot form a gel and therefore cannot be used for gel testing.

[0202] It should be noted that while the preferred embodiments of the present invention are provided in this specification, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are not intended to impose additional limitations on the content of the present invention; their purpose is to provide a more thorough and comprehensive understanding of the disclosure of the present invention. Furthermore, the above-described technical features can be combined with each other to form various embodiments not listed above, all of which are considered to be within the scope of the present invention. Moreover, those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A bone repair implant based on a cold-cured resin glycerol phosphate calcium composite gel, characterized in that, The bone repair implant comprises components A, B, and C, wherein component A is a cold-curing resin, component B is sodium hyaluronate, and component C is calcium glycerophosphate, magnesium citrate, calcium gluconate, and calcium amino acids, wherein components A, B, and C form a composite gel. The preparation method of the bone repair implant includes: 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 until homogeneous; allowing it to stand to form a gel; and then freeze-drying to obtain the final product. The aqueous solutions of components A, B, and C are mixed in a volume ratio of 1.0-2.0:1.0-2.0:0.5-2.

5. The concentration of the aqueous solution of component A is 1.0-20 g / L; The concentration of the aqueous solution of component B is 5.0-20.0 g / L; 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 gluconate, and calcium amino acids 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; solutions I and II are mixed in a volume ratio of 1.0:0.5-2.5 to obtain the aqueous solution of component C, wherein the mass ratio of magnesium citrate, calcium gluconate, and calcium amino acids is 10-25:20-50:70-150.

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; The high molecular weight sodium hyaluronate has a molecular weight range of 1,800,000 to 2,200,000, and the medium molecular weight sodium hyaluronate has a molecular weight range of 1,000,000 to 1,800,000. The mass ratio of high molecular weight sodium hyaluronate to medium molecular weight sodium hyaluronate is 0.5-2:2-0.

5.

3. The bone repair implant according to claim 1, characterized in that, Calcium glycerophosphate includes one or more of β-calcium glycerophosphate, D(+)α-calcium glycerophosphate, and L(-)α-calcium glycerophosphate; Calcium gluconate includes one or both of calcium gluconate and L-threonate calcium; Amino acid calcium includes one or more of the following: glycine calcium, lysine calcium, L-aspartic acid calcium, aspartic acid calcium, glutamate calcium, and L-histidine calcium.

4. A method for preparing a bone repair implant based on a cold-cured resin glycerophosphate calcium composite gel according to any one of claims 1-3, characterized in that, The process includes preparing aqueous solutions of components A, B, and C separately, first mixing aqueous solutions of components A and B, then adding aqueous solution of component C, stirring and mixing thoroughly, allowing it to stand to form a gel, and then freeze-drying to obtain the bone repair implant based on the cold-cured resin glycerol phosphate calcium composite gel. The aqueous solutions of components A, B, and C are mixed in a volume ratio of 1.0-2.0:1.0-2.0:0.5-2.

5. The aqueous solution of component A is prepared as follows: the cold-curing resin is dissolved 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 later use; 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 later use; 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 gluconate, and calcium amino acids 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; solutions I and II are mixed in a volume ratio of 1.0:0.5-2.5 to obtain the aqueous solution of component C, which is then stored below 10°C for later use. The mass ratio of magnesium citrate, calcium gluconate, and calcium amino acids is 10-25:20-50:70-150.

5. The preparation method according to claim 4, characterized in that, The dissolution is carried out at 0-80°C under ultrasonic or stirring conditions.

6. The preparation method according to claim 4, characterized in that, The dissolution of calcium glycerophosphate was carried out at below 20°C under ultrasound or stirring; the dissolution of magnesium citrate, calcium gluconate, and calcium amino acids was carried out at room temperature under ultrasound or stirring.

7. The preparation method according to claim 4, characterized in that, The gel formation process involves first heating a mixed aqueous solution of components A, B, and C to 50-80°C under nitrogen protection and holding it for 15-45 minutes, then transferring it to a mold and cooling it to room temperature for 2-10 hours.

8. The preparation method according to claim 4, characterized in that, The preparation method further includes sterilizing the aqueous solution of the mixed components A, B and C before mixing them.

9. The preparation method according to claim 8, characterized in that, The sterilization process is ethylene oxide sterilization.

10. The preparation method according to claim 4, characterized in that, The preparation method also includes slicing or cutting the formed gel before freeze-drying to obtain the desired shape and size.

11. The use of the bone repair implant based on the cold-cured resin glycerophosphate calcium composite gel according to any one of claims 1-3 or the bone repair implant based on the cold-cured resin glycerophosphate calcium composite gel prepared by the preparation method according to any one of claims 4-10 in the preparation of materials for the repair of cancellous bone and / or cartilage.

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