Bone bonding material with high strength and low immunoreaction as well as preparation method and application method of bone bonding material

Abstract

The invention discloses a high-strength low-immunoreaction bone bonding material and a preparation and application method thereof. The bone binding material comprises tetracalcium phosphate, serine phosphate, magnesium phosphate and a sodium citrate solution. According to the preparation method, tetracalcium phosphate, phosphoserine, magnesium phosphate powder and a sodium citrate solution are fully mixed and reacted by utilizing a hedging device consisting of a screw injector and a connecting pipe. And extruding the mixed liquid material through a filter membrane to complete the preparation of the bone adhesive material. The bone binding material can be used for preparing bionic sclerotin, binding broken bones in comminuted fractures or smearing the bone binding material to bone gaps, and in the decomposition process, the bone binding material can continuously release magnesium ions, so that the immune environment of early bone repair is regulated and controlled, the bone repair process is accelerated, and bone healing is promoted. Meanwhile, experimental data prove that the bone adhesive material provided by the invention can provide stable supporting strength, has no potential toxicity to fibroblasts, and is good in biocompatibility.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical materials, relates to a bone adhesive material and its preparation and application methods, and particularly relates to a high-strength and low-immune-response bone adhesive material and its preparation and application methods. Background Art

[0002] The treatment of comminuted fractures is a difficult problem in clinical treatment. Since the broken bones are difficult to be effectively reduced and fixed, they can only be discarded, resulting in bone defects causing nonunion, and reducing the success rate and rate of bone healing.

[0003] Bone adhesives can form a strong adhesion at the fracture ends to restore the physiological structure of the bone and ensure the support and stress strength. Prior art 1 (CN202310767115.2) discloses an injectable fracture healing adhesive, which mimics the characteristics of the side-chain carboxyl groups of marine barnacle biomaterials and shows good adhesion effect. However, its composition is quite different from that of bone tissue, and there are still problems in mechanical strength and biological safety. Prior art 2 (CN202211370224.2) discloses a photo-curable bone adhesive, but it requires light curing during use, and the convenience of use is relatively low.

[0004] Artificial bone materials can mimic the natural bone tissue structure, thus promoting bone ingrowth and integration. Prior art 3 (CN200910043405.2) and prior art 4 (CN202210656568.3) disclose a preparation method of a bionic bone material with non-uniform pore distribution and a bionic bone material based on icariin-functionalized polylactic acid and its preparation method. However, there are significant differences in material composition and physical properties from natural bone tissue, and it is difficult to achieve immediate bone integration. Prior art 5 (CN202410688887.1) discloses a method for controlling the solidification time of bionic bone by adjusting the composition of the reaction liquid phase. During the preparation process of the material, a counterflush form is adopted to promote the complete fusion and reaction of the reaction liquid and the solid phase. However, a large number of bubbles will be introduced during the counterflush process, affecting the adhesion strength and stability of the material.

[0005] The immune environment is directly related to the bone repair process. Therefore, in addition to considering the compositional similarity between the material and natural bone tissue as well as the adhesion strength and stability, it is also necessary to consider the impact of the implantation and degradation processes of the material on the immune environment of the repair site. The artificial materials mentioned in the above prior arts will continuously cause inflammatory reactions at the implantation site due to the metabolic products generated during the bone healing process, resulting in difficulty in healing the defect site. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides a bone adhesive material with high strength and low immune response, as well as its preparation and application methods. After the raw materials are subjected to counter-jet homogenization and membrane treatment, a bone adhesive material with high density, high strength, low porosity, and low toxicity is obtained. It can bond fragmented bones and provide stable support strength, and can also regulate the immune environment of early bone repair, accelerate the bone repair process, promote bone healing, and solve the problem of reduction of small fragmented bones in comminuted fractures.

[0007] A bone adhesive material with high strength and low immune response, comprising tetracalcium phosphate, phosphoserine, magnesium phosphate, and sodium citrate solution. The mass ratio of tetracalcium phosphate, phosphoserine, magnesium phosphate, and sodium citrate solution is: 1 g: 0.5 g: (39.45 - 78.9) mg: 0.35 g.

[0008] Preferably, the concentration of the sodium citrate solution is 0.223 g / mL.

[0009] A preparation method of a bone adhesive material with high strength and low immune response, specifically comprising the following steps:

[0010] Step 1: Mix the powders of tetracalcium phosphate, phosphoserine, and magnesium phosphate according to the mass ratio of 1 g: 0.5 g: (39.45 - 78.9) mg, and load them into syringe I with a screw cap.

[0011] Step 2: Load the sodium citrate solution with a concentration of 0.223 g / mL into syringe II with a screw cap.

[0012] Step 3: Connect syringe I and II with a connecting tube, and push the sodium citrate solution in syringe II into syringe I through the connecting tube.

[0013] Step 4: Push the mixture in syringe I into syringe II.

[0014] Step 5: Repeat multiple times to make the powder and solution evenly mixed, and then extrude the mixed solution through a filter membrane to obtain the bone adhesive material.

[0015] Preferably, the pore size of the extrusion membrane is 1 - 100 μm.

[0016] Preferably, the pore size of the extrusion membrane is 5 μm, 10 μm, 30 μm, or 100 μm.

[0017] Preferably, the material of the extrusion membrane is PES, PTFE, PVDF, or nylon.

[0018] Application of a bone adhesive material with high strength and low immune response in the preparation of biomimetic bone mass.

[0019] Preferably, use the bone adhesive material to bond the fragmented bones in comminuted fractures.

[0020] Preferably, apply the bone adhesive material to the bone gap and let it solidify for 5 minutes.

[0021] The present invention has the following beneficial effects:

[0022] 1. During the degradation process of the bone adhesive material, magnesium ions are slowly released, which participate in the regulation of the immune environment at the repair site, inhibit the excessive release of pro-inflammatory cytokines and promote the production of anti-inflammatory factors, and reduce the production of reactive oxygen species (ROS) by immune cells under stress through the antioxidant effect of magnesium ions.

[0023] 2. The bone adhesive material obtained through countercurrent preparation and membrane treatment has a strong adhesive effect, can firmly bond bone to bone and bone to metal, and forms a bionic bone mass through reaction, which is conducive to immediate integration with autologous bone in clinical applications.

[0024] 3. Through membrane extrusion, the negative impact of magnesium phosphate on the tensile strength of the bone adhesive material can be offset, and even the tensile strength and torsional strength of the bone adhesive material can be further improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the scanning electron microscope image of the bone adhesive material in Test Example 2;

[0026] Figure 2 It is the EDS elemental analysis result of the bone adhesive material in Test Example 2;

[0027] Figure 3 It is the schematic diagram of the pore distribution of the bone adhesive material in Test Example 2;

[0028] Figure 4 It is the fluorescence quantitative amplification result in Test Example 4;

[0029] Figure 5 It is the fracture model and adhesion test result in Test Example 5. DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention will be further explained below with reference to the accompanying drawings;

[0031] Example 1

[0032] This example provides a preparation method of a bone adhesive material with high strength and low immune response.

[0033] Weigh 1.0 g of tetracalcium phosphate and 0.5 g of phosphoserine, mix them evenly and load them into a 10 mL screw-cap syringe I. Load 0.35 mL of a sodium citrate solution with a concentration of 0.223 g / mL into a 5 mL screw-cap syringe II. Connect the screw-cap syringes I and II with a connecting tube. Push the sodium citrate solution in syringe II into syringe I through the connecting tube, and push it back and forth repeatedly to make the powder and the fusion mixture evenly mixed, obtaining a pasty bone adhesive material.

[0034] Example 2

[0035] This example provides a method for preparing a bone adhesive material with high strength and low immune response. The difference from Example 1 is that 1.0 g of tetracalcium phosphate, 0.5 g of phosphoserine and 39.45 mg of magnesium phosphate are weighed, evenly mixed and loaded into a 10 mL screw-cap syringe I.

[0036] Example 3

[0037] This example provides a method for preparing a bone adhesive material with high strength and low immune response. The difference from Example 2 is that the weight of the magnesium phosphate is 78.9 mg.

[0038] Example 4

[0039] This example provides a method for preparing a bone adhesive material with high strength and low immune response. The difference from Example 3 is that for the mixed solution, it is extruded through PES filter membranes with pore sizes of 5 μm and 10 μm respectively to make a pasty bone adhesive material.

[0040] Example 5

[0041] This example provides a method for preparing a bone adhesive material with high strength and low immune response. The difference from Example 3 is that for the mixed solution, it is extruded through PTFE filter membranes with pore sizes of 10 μm and 30 μm respectively to make a pasty bone adhesive material.

[0042] Example 6

[0043] This example provides a method for preparing a bone adhesive material with high strength and low immune response. The difference from Example 3 is that for the mixed solution, it is extruded through nylon filter membranes with pore sizes of 10 μm and 100 μm respectively to make a pasty bone adhesive material.

[0044] Test Example 1

[0045] For the bone adhesive materials prepared in Examples 1 - 6, tests on compressive strength and tensile strength were carried out:

[0046] Step 1: Extrude the bone adhesive materials prepared in Examples 1 - 6 into a mold with a diameter of 6 mm for curing, take out the cured bone adhesive materials, and conduct compressive tests.

[0047] Step 2: Evenly apply the bone cement materials prepared in Examples 1 - 6 on both ends of the broken ends of porcine femurs. After curing, place the bonded porcine femurs in a fixture and measure the tensile strength. The direction of the applied force is perpendicular to the surface of the bonded porcine femurs. Connect the testing machine and the bonded porcine femurs using a fixture, and test the tensile strength of the bonded porcine femurs at a speed of 20 mm / min.

[0048] Step 3: Squeeze the bone cement materials prepared in Examples 1 - 6 into a module of 1 cm * 1 cm size and insert an implant screw. After curing, use a torque meter to detect the torque of the screw.

[0049] The results are shown in Table 1:

[0050] Table 1

[0051]

[0052] It can be seen from the results in Table 1 that the addition of magnesium phosphate will slightly reduce the tensile strength of the bone cement material. However, after extrusion through membranes with different pore sizes, it can be observed that the tensile strength of the bone cement material increases significantly, especially when extruded through a 100 - μm nylon membrane, which is the most significant. Similar results can also be seen in the torque of the bone cement material on the screw. The addition of magnesium phosphate and membrane extrusion have little effect on the compressive strength of the bone cement material.

[0053] Test Example 2

[0054] Use a scanning electron microscope to observe the bone cement materials extruded through a 100 - μm pore - sized nylon filter membrane in Examples 1, 3, and 6, and observe the surface conditions of the materials, as Figure 1 shown, the surface structures of the bone cement materials obtained in Examples 1, 3, and 6 have not changed significantly, indicating that the addition of magnesium phosphate does not cause a significant impact on the surface structure of the bone cement material. For the bone cement material extruded through the membrane in Example 6, compared with the bone cement materials not extruded through the membrane in Examples 1 and 3, the pores on the surface are significantly reduced and it is smoother.

[0055] Conduct EDS elemental analysis on the bone cement materials extruded through a 100 - μm pore - sized nylon filter membrane in Examples 1, 3, and 6. The proportion of each element in the material is as Figure 2 shown, after adding magnesium ions, the content of magnesium ions in the bone cement material increases while the ratio of calcium ion content decreases. However, there is not much difference before and after membrane filtration.

[0056] Observe the pore size, proportion of pore area, and proportion of pore volume of the bone cement materials extruded through a 100 - μm pore - sized nylon filter membrane in Examples 1, 3, and 6, as Figure 3As shown, the addition of magnesium phosphate and passing through the membrane do not significantly change the internal micro-porosity size of the bone adhesive material. However, the bone adhesive materials prepared in Examples 1 and 3 are prone to generating voids with a diameter of 1-2 mm, which affects the internal bonding strength after final curing. The bone adhesive material after membrane extrusion in Example 6 has the characteristics of being delicate and dense.

[0057] Test Example 3

[0058] Perform biocompatibility tests on the bone adhesive materials extruded through a 100-μm pore size nylon filter membrane in Examples 1, 3, and 6:

[0059] Step 1: Add the bone adhesive material to the complete medium at 0.2 g / mL and extract it at 37°C for 72 hours to obtain an extract.

[0060] Step 2: Adjust the concentration of fibroblast L929 cultured to the logarithmic growth phase to 10 6 cells / L and evenly seed them in a 96-well plate, 100 μL per well. After culturing at 37°C for 24 hours, remove the medium and add 100% extract, 75% extract, 50% extract, 25% extract, and complete medium respectively, 100 μL per well.

[0061] Step 3: After continuing to culture at 37°C for 24 hours, remove the medium, add 50 μL per well, and incubate at 37°C for 2 hours.

[0062] Step 4: Add 100 μL of isopropanol to each well, and use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance value at a wavelength of 570 nm, so as to calculate the survival rate of fibroblasts added with the extract relative to the complete medium, in order to measure the cytotoxicity of the bionic bone quality to fibroblasts. The results are shown in Table 2 below:

[0063] Table 2

[0064] Example 100% extract 75% extract 50% extract 25% extract 1 89％ 96％ 98％ 102％ 3 81％ 91％ 89％ 128％ 6 83％ 92％ 94％ 110％

[0065] As can be seen from Table 2, for different contents of magnesium phosphate, the survival rate of fibroblasts under 100% extract is greater than 70%. It can be considered that the bone adhesive material has no potential toxicity to fibroblasts and has good biocompatibility.

[0066] Test Example 4

[0067] Perform immunomodulatory activity tests on the bone adhesive material extruded through a 100-μm pore size nylon filter membrane in Example 6:

[0068] Step 1: Add the bone adhesive material to the complete medium at 0.2 g / mL and extract it at 37°C for 72 hours to obtain an extract.

[0069] Step 2: Mouse primary macrophages from the femur marrow of 6 - 8 week-old C57BL / 6 mice were cultured in α-MEM medium containing 25 ng / ml M-CSF and 10% serum to induce them into macrophages.

[0070] Step 3: The macrophages were evenly seeded into a 12-well cell culture plate, with 200,000 cells per well.

[0071] Step 4: The cells were divided into a control group, an LPS group, an extract group, and an LPS + extract group.

[0072] The culture medium was removed. 1 mL of complete medium was added to the control group, complete medium containing 100 μg / mL LPS was added to the LPS group, 1 mL of biomimetic bone matrix extract was added to the extract group, and 1 mL of biomimetic bone matrix extract containing 100 μg / mL LPS was added to the LPS + extract group.

[0073] Step 5: After culturing at 37°C for 6 hours, the cells were collected, RNA was extracted using TriZol and reverse transcribed into cDNA, and the target gene was subjected to fluorescence quantitative amplification using primers for inflammation-related genes. The results are as Figure 4 shown. It can be seen that the bone adhesive material prepared in Example 6 can significantly reduce the expression of the pro-inflammatory factor-related gene IL-1α in LPS-stimulated macrophages and increase the expression of the anti-inflammatory factor-related gene YM-1, indicating that it can reduce the pro-inflammatory ability of macrophages.

[0074] Test Example 5

[0075] The bone adhesive material extruded through a 100 μm pore size nylon filter membrane in Example 6 was used for the adhesion test on a fracture model:

[0076] Step 1: After anesthetizing the rabbit, the skin of the leg was shaved, disinfected, and incised, and the femoral shaft was exposed layer by layer. A comminuted fracture of the shaft was formed using a saw to complete the modeling of a circular defect in the femoral condyle of the rabbit.

[0077] Step 2: After fixing the main bone shaft with a steel plate, the bone fragments were adhered using the bone adhesive material prepared in Example 6, as Figure 5 shown. It can be seen that the bone adhesive material can well adhere the bone fragments after curing, thereby reducing the bone defect caused by comminuted fracture.

Claims

1. A high-strength, low-immunoreactive bone adhesive material, characterized in that: It includes tetracalcium phosphate, phosphoserine, magnesium phosphate and sodium citrate solution; the mass ratio of the tetracalcium phosphate, phosphoserine, magnesium phosphate and sodium citrate solution is: 1g:0.5g:(39.45-78.9)mg:0.35g; the material can provide stable mechanical strength and participate in the immune environment regulation of the repair site during the degradation process.

2. A high-strength, low-immune-reaction bone adhesive material as claimed in claim 1, characterized in that: The concentration of the sodium citrate solution is 0.223 g / mL.

3. A method for preparing a high-strength, low-immunoreactive bone adhesive material, characterized in that: The specific steps include: Step 1, tetracalcium phosphate, phosphoserine, and magnesium phosphate powders are mixed in a mass ratio of 1 g: 0.5 g: (39.45-78.9) mg and loaded into a screw-mouth syringe I; Step 2, loading a sodium citrate solution having a concentration of 0.223 g / mL into a screw-mouth syringe II; Step 3, use a connecting tube to connect the screw-mouth syringes I and II, and push the sodium citrate solution in the screw-mouth syringe II into the screw-mouth syringe I through the connecting tube; Step 4, push the mixture in the screw-mouth syringe I into the screw-mouth syringe II; Step 5: repeat several times to make the powder and solution evenly mixed, and then extrude the mixed solution through a filter membrane to obtain the bone adhesive material.

4. The method for preparing a high-strength low-immunoreactive bone adhesive material as claimed in claim 3, characterized in that: The pore size of the extruded membrane is 1 to 100 μm.

5. A method for preparing a high-strength low-immunoreactive bone adhesive material as claimed in claim 3 or 4, characterized in that: The pore size of the extruded membrane is 5 μm, 10 μm, 30 μm or 100 μm.

6. A method for preparing a high-strength low-immunoreactive bone adhesive material as claimed in claim 3 or 4, characterized in that: The material of the extruded film is PES, PTFE, PVDF or nylon.

7. The method for preparing a high-strength and low-immunoreactive bone adhesive material as claimed in claim 3, characterized in that: The volume of the screw-mouth syringe I is 10 mL, and the volume of the screw-mouth syringe II is 5 mL.

8. Application of a high-strength, low-immunoreactive bone adhesive material, characterized in that: Use of the bone adhesive material as claimed in claims 1 to 4 in the preparation of bionic bone, or in bonding broken bones in comminuted fractures.

9. The use of a high-strength and low-immunoreactive bone adhesive material as claimed in claim 8, characterized in that: The bone adhesive material is applied to the bone gap and solidified for 3 to 10 minutes.

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