Bone cement as well as preparation method and application thereof

Abstract

The invention relates to the field of biomedical materials, in particular to bone cement as well as a preparation method and application thereof. Comprising the steps that a component A, a component B and a component C react to form a citrate-based prepolymer, the component A is citric acid polyester containing at least two reactive ester-based functional groups on a terminal group or a side chain, the component B is non-citric acid polyester containing at least two reactive functional groups on a terminal group or a side chain, and the functional groups are the same or different; the functional groups are selected from at least one of ester groups, hydroxyl groups, amino groups, sulfydryl groups, epoxy groups, carboxyl groups, acid anhydrides and organic substituent groups; the component C is polyol; the reaction comprises an ester exchange reaction and / or a condensation polymerization reaction and a substitution reaction; the injectable citrate bone cement is prepared by blending the citrate-based prepolymer, isocyanate and an additive, has the advantages of low curing temperature, degradability, excellent mechanical properties and osteogenesis promotion, and is suitable for orthopedics, brain surgery and cranio-maxillofacial plastic repair.

Description

A bone cement, its preparation method, and its uses Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a bone cement, its preparation method, and its uses. Background Technology

[0002] Bone cement is a self-curing biomaterial used to fill gaps or cavities between bone and implants. Its injectability allows for effective filling and repair of irregular bone injuries, and its rapid curing provides good mechanical strength, significantly shortening surgical time. To date, polymethyl methacrylate (PMMA), calcium phosphate, and magnesium phosphate bone cements have been widely used in orthopedics. Among them, PMMA has become the most clinically used material due to its excellent mechanical properties, good processability, and low complication rate. However, this material still has some drawbacks, including bioinertness, non-degradability, potential toxicity, and exothermic polymerization (temperatures exceeding 60°C), which limit its clinical application. Studies have shown that bone tissue necrosis occurs within one minute at temperatures above 50°C, which also affects the mechanical interlocking effect between bone and PMMA. Therefore, developing injectable bone cement materials with good bioactivity, biodegradability, and high performance has significant clinical value and urgent research significance.

[0003] Patent document CN113855855B discloses a PMMA bone cement and its preparation method. This patent adds a modified cashew nut shell monomer with antibacterial properties and phase change microspheres to the reaction system, giving the bone cement good antibacterial properties, a low curing temperature, and excellent mechanical properties (90 MPa). However, its curing time is 25-35 minutes, longer than the 5-15 minutes specified in the national standard ISO5833:2002, which could prolong the surgical time.

[0004] Patent document CN1208095C discloses a method for preparing medical composite bone cement powder and bone cement liquid. This patent uses a blend of soda lime and polyamide as bone cement powder, and a calcium chloride ethanol solution as bone cement liquid, which are then compounded in a specific ratio to obtain an injectable bone cement material. The bone cement prepared by this method has ideal curing properties and minimal temperature change; however, its maximum compressive strength is only 37 MPa, far lower than the 100 MPa strength of PMMA bone cement.

[0005] Patent document CN105396175B discloses a method for preparing and applying bone cement containing calcium citrate. This invention is the first to combine calcium citrate and calcium phosphate salts to form bone cement. The prepared bone cement exhibits good plasticity, osteoconductive properties, osteoinductive activity, cell activity, and a relatively good degradation rate, making it suitable for orthopedics, neurosurgery, and craniofacial plastic surgery repair. However, its final setting time is greater than 15 minutes, and its compressive strength is less than 45 MPa, failing to meet the national standard ISO 5833:2002 requirement of greater than 70 MPa.

[0006] In summary, traditional PMMA bone cement still faces numerous challenges in its application. To address these issues, many alternatives to PMMA components have been developed, demonstrating significant advantages in lowering polymerization temperature, improving biocompatibility, and enhancing degradability. However, their mechanical strength is generally inferior to that of PMMA bone cement. Therefore, developing a bone cement that can completely replace PMMA components while possessing high compressive strength, biodegradability, injectability, and good biocompatibility has become a key direction in current bone cement research. Summary of the Invention

[0007] The technical problem to be solved by this invention is to overcome the problems of toxicity, high exothermic temperature, and mismatch of mechanical properties in existing bone cement materials, thereby providing a bone cement, its preparation method, and its uses. The bone cement is a citrate-based bone cement, a bio-based injectable material with fast curing time, low curing temperature, and good biocompatibility, which can reduce the difficulty of surgical operation. Moreover, the bone cement material is biodegradable and its mechanical properties are comparable to PMMA, exhibiting good osteogenic properties, and is suitable for orthopedics, neurosurgery, and craniofacial plastic and reconstructive surgery.

[0008] In a first aspect, the present invention provides a method for preparing bone cement, comprising: reacting components A, B, and C to form a citrate-based prepolymer; component A being a citrate polyester containing at least two reactive ester functional groups on its end groups or side chains; component B being a non-citrate polyester containing at least two reactive functional groups on its end groups or side chains, wherein the functional groups are the same or different; the functional groups are selected from at least one of ester groups, hydroxyl groups, amino groups, mercapto groups, epoxy groups, carboxyl groups, acid anhydrides, and organic substituents; component C being a polyol; the reaction comprising an ester exchange reaction; or a combination of an ester exchange reaction and at least one of a condensation reaction and a substitution reaction; taking the citrate-based prepolymer and isocyanate to prepare bone cement, and performing a crosslinking reaction of urethane bonds or amide bonds.

[0009] In some embodiments, the citrate polyester is at least one selected from dimethyl citrate, trimethyl citrate, diethyl citrate, triethyl citrate, dipropyl citrate, or tributyl citrate; and / or, component B is a reinforcing polyester selected from at least one selected from phthalate, dioctyl adipate, dioctyl sebacate, isooctyl mercaptoacetate, methyl 3-aminoacrylate, pentaerythritol ester, or diethyl tartrate; and / or, the polyol is at least one selected from butanediol, octyl glycol, bis(4-hydroxyphenyl) disulfide, resveratrol, bis(2-hydroxyethyl) disulfide, isosorbide, polyethylene glycol, xylitol, citric acid, or N-methyldiethanolamine.

[0010] In some embodiments, the step of forming the citrate-based prepolymer further includes a catalyst, wherein the catalyst is at least one of stannous octoate, dibutyltin dilaurate, or zinc chloride.

[0011] In some embodiments, the isocyanate is selected from at least one of isophorone diisocyanate, 1,6-hexamethylene diisocyanate, toluene diisocyanate, naphthalene diisocyanate, 1,4-cyclohexyl diisocyanate, or 4,4'-diphenylmethane diisocyanate.

[0012] In some embodiments, the preparation of bone cement also includes additives selected from bone-promoting additives, contrast agents, or antibacterial agents; the bone-promoting additives are selected from at least one of hydroxyapatite, magnesium hydroxide, calcium phosphate, inorganic ceramics, plexiglass, alendronate, or clay; the contrast agent is selected from at least one of zirconium oxide, barium sulfate, strontium polyphosphate, strontium salts, graphene-iodohydrin, or tungsten / bismuth oxide; and the antibacterial agent is selected from at least one of antibiotics, antimicrobial peptides, inorganic metal ions, or quaternary ammonium salts.

[0013] In some embodiments, the following raw materials are included, by weight parts: 1-15 parts by weight of citric acid polyester, 1-15 parts by weight of component B, 2-30 parts by weight of polyol, and 0.001-2 parts by weight of catalyst; wherein the weight parts of the polyol are greater than or equal to the sum of the weight parts of citric acid polyester and reinforcing polyester; wherein the isocyanate is 0.1-100 parts, and the osteogenic additive is 0-10 parts.

[0014] In some embodiments, the reaction conditions in the step of forming the citrate-based prepolymer are a temperature of 140-180°C and a reaction time of 24-48 h.

[0015] In some embodiments, the conditions for the crosslinking reaction of the urethane or amide bonds are: room temperature, curing time of 0-20 min, and reaction time > 0 min, and injectability before complete curing; the exothermic temperature during curing is 0-45°C, and ≠ 0°C.

[0016] Secondly, embodiments of the present invention provide a bone cement prepared by the bone cement preparation method described above; optionally, the compressive strength of the bone cement is 0~200 MPa and the compressive modulus is 0~2 GPa.

[0017] Thirdly, embodiments of the present invention provide the use of the bone cement described herein in the preparation of orthopedic, neurosurgical, or craniofacial plastic and repair materials.

[0018] The technical solution of this invention has the following advantages: 1. The present invention provides a method for preparing bone cement, comprising: reacting components A, B, and C to form a citrate ester-based prepolymer; component A is a citrate polyester containing at least two reactive ester functional groups on its end groups or side chains; component B is a non-citrate polyester containing at least two reactive functional groups on its end groups or side chains, wherein the functional groups are the same or different; the functional groups are selected from at least one of ester groups, hydroxyl groups, amino groups, mercapto groups, epoxy groups, carboxyl groups, acid anhydrides, and organic substituents; component C is a polyol; the reaction includes transesterification reaction, or a combination of transesterification reaction and at least one of condensation reaction and substitution reaction; taking the citrate ester-based prepolymer and isocyanate to prepare bone cement, and performing a crosslinking reaction of urethane bonds or amide bonds. In the above scheme, since citric acid is a highly crystalline monomer, it needs to be melted at high temperature. Therefore, the citrate ester-based prepolymer prepared from citric acid has high viscosity and poor injectability, and often requires organic solvents for dissolution. In this invention, the citric acid-based prepolymer prepared by using hydrophilic monomers such as polyols as chain extenders is soluble in water, but it reacts with isocyanates and releases heat violently during the curing process. When used as an injectable filler before curing, it damages the surrounding tissue. However, the citric acid ester-based prepolymer prepared from liquid citric acid polyester is liquid, requiring no additional solvent for dissolution, and possesses excellent injectability. This effectively lowers the curing temperature, reduces damage to surrounding tissues, significantly simplifies surgical procedures, and improves the success rate. Furthermore, the bone cement material exhibits satisfactory curing time, degradation rate, mechanical strength, and tissue compatibility. The prepared citric acid ester-based material is biodegradable in vivo, and the exogenous citric acid released during degradation can promote the repair of defect sites through metabolic regulation. In summary, compared with traditional PMMA bone cement materials, the citric acid ester-based bone cement material prepared in this invention has lower production costs, simpler preparation methods, better biocompatibility, and mechanical properties comparable to PMMA bone cement, meeting the mechanical performance requirements of load-bearing bone components. The biodegradable material simplifies surgical procedures, avoids the need for removal of PMMA bone cement, and improves the success rate of surgery. Furthermore, this material has multifunctional applications and can be used as a carrier material to load various additives, such as promoting bone growth, in vivo imaging, antibacterial, and antitumor functions. This bio-based bone cement material has broad prospects for transformation and application in the field of bone defect repair.

[0019] 2. This invention provides a method for preparing bone cement, comprising an isocyanate selected from at least one of isophorone diisocyanate, 1,6-hexamethylene diisocyanate, toluene diisocyanate, naphthalene diisocyanate, 1,4-cyclohexyl diisocyanate, or 4,4'-diphenylmethane diisocyanate. Curing is achieved through one-step crosslinking of the isocyanate, and the crosslinking and curing speed can be controlled by varying the amount of crosslinking agent added, enabling multiple preparation possibilities such as 3D printing, injection molding, and mold casting, thus broadening the possibilities for material transformation and application. Furthermore, toluene diisocyanate and naphthalene diisocyanate have rigid molecular structures, resulting in bone cement with stronger mechanical properties.

[0020] 3. The present invention provides a method for preparing bone cement, during which various additives can be added as needed without affecting the original performance during the curing process. These additives include osteogenic additives, selected from at least one of hydroxyapatite, magnesium hydroxide, calcium phosphate, inorganic ceramics, plexiglass, alendronate, or clay; contrast agents, selected from at least one of zirconium oxide, barium sulfate, strontium polyphosphate, strontium salts, graphene-iodohydrin, or tungsten / bismuth oxide; and bactericides, selected from at least one of antibiotics, antimicrobial peptides, inorganic metal ions, or quaternary ammonium salts. This bio-based bone cement material is biodegradable and releases corresponding additives during degradation, enabling multifunctional treatment. The accompanying drawings are provided to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art. The drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 is a schematic diagram of the citrate-based bone cement prepared according to the steps of Example 1. The left figure is an effect diagram of the liquid citrate prepolymer prepared according to step (1) of Example 1, and the right figure is an effect diagram of the citrate bone cement prepared according to step (2) of Example 1.

[0022] Figure 2 shows the injection performance of the citrate-based bone cement prepared according to Example 2.

[0023] Figure 3 shows the mechanical properties of the citrate-based bone cement prepared according to Examples 1 and 2.

[0024] Figure 4 shows the curing time of the citrate-based bone cement prepared according to the steps of Examples 2 and 3.

[0025] Figure 5 is a curing temperature image of the citrate-based bone cement prepared according to the steps of Examples 2 and 3.

[0026] Figure 6 is an image showing the compressive strength of the citrate-based bone cement prepared according to the steps of Example 3.

[0027] Figure 7 is an ALP staining image of the citrate-based bone cement prepared according to the steps of Examples 2 and 3.

[0028] Figure 8 shows the compressive strength of the citrate-based bone cement prepared according to the steps of Examples 4-8.

[0029] Figure 9 is a surface modulus image of the citrate-based bone cement prepared according to the steps of Examples 5-8.

[0030] Figure 10 shows the degradation rate of the citrate-based bone cement prepared according to Examples 6, 9 and 10.

[0031] Figure 11 is a picture of the citric acid release from the citrate-based bone cement prepared according to Examples 6, 9 and 10. The left picture shows the citric acid content released into the supernatant, and the right picture shows the citric acid content in the cells after co-incubation with cells.

[0032] Figure 12 is a cross-sectional SEM image of the fully cured citrate-based bone cement prepared according to Example 11.

[0033] Figure 13 shows images of the angiogenic properties of the citrate-based bone cement prepared according to Examples 8 and 11.

[0034] Figure 14 is a cell image of the surface of the citrate-based bone cement prepared according to Examples 8, 9 and 11.

[0035] Figure 15 shows the blood compatibility of the citrate-based bone cement prepared according to Examples 8-11.

[0036] Figure 16 is an image of the antibacterial zone of the citrate-based bone cement prepared according to Examples 1 and 12.

[0037] Figure 17 is a stained image of citrate-based bone cement prepared according to Examples 6 and 13, co-cultured with macrophages.

[0038] Figure 18 is an RNA sequencing diagram of the fully cured citrate-based bone cement prepared according to Example 13. The left diagram is a volcano diagram, and the right diagram shows differentially regulated genes.

[0039] Figure 19 shows the in vivo curing of the fully cured citrate bone cement prepared according to Example 13.

[0040] Figure 20 shows the curing time of citrate bone cement prepared according to the preparation methods of Comparative Example 1 and Comparative Example 2.

[0041] Figure 21 is a picture of the citrate bone cement prepared according to the preparation method of Comparative Example 3 after pressure testing.

[0042] Figure 22 shows the curing temperature of the citrate bone cement prepared according to the preparation method of Comparative Example 4.

[0043] Figure 23 is a picture of the citrate bone cement prepared according to the preparation method of Comparative Example 5 after pressure testing.

[0044] Figure 24 is a physical image of the citrate prepolymer prepared according to the preparation methods of Comparative Example 6 and Example 10. The left image is Comparative Example 6, and the right image is Example 10. Detailed Implementation

[0045] The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0046] Unless otherwise specified, all experimental steps or conditions in the examples were performed according to conventional experimental procedures and conditions in the art. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0047] Example 1 (1) 8 g of trimethyl citrate, 1 g of dioctyl adipate, 10 g of butanediol and 0.05 g of stannous octoate were added to a flask and reacted under vacuum at 160°C for 36 h. The reactants were precipitated with deionized water and obtained by freeze drying. (2) 1 g of the citrate prepolymer was weighed and added to a polytetrafluoroethylene (PTFE) mold. 0.3 g of 1,6-hexamethylene diisocyanate was added and the two components were stirred evenly. The mixture was allowed to stand at room temperature for 5-15 minutes (the complete curing time in this example was 8.07 min) to obtain citrate bone cement.

[0048] Figure 1 is a schematic diagram of the citrate-based bone cement prepared according to the steps of Example 1. The left figure is an effect diagram of the liquid citrate prepolymer prepared according to step (1) of Example 1, and the right figure is an effect diagram of the citrate bone cement prepared according to step (2) of Example 1.

[0049] Example 2 (1) 8 g of trimethyl citrate, 1 g of dioctyl adipate, 10 g of butanediol and 0.05 g of stannous octoate were added to a flask and reacted under vacuum at 160°C for 36 h. The reactants were precipitated with deionized water and obtained by freeze drying. (2) 1 g of the citrate prepolymer was weighed and added to a polytetrafluoroethylene (PTFE) mold. 0.3 g of toluene diisocyanate was added and the two components were stirred evenly. The mixture was allowed to stand at room temperature for 5-15 minutes (the complete curing time in this example was 8.44 min) to obtain citrate bone cement.

[0050] Figure 2 shows the injection performance of the citrate-based bone cement prepared according to Example 2. The bone cement prepared by compounding with the crosslinking agent was filled into a syringe and pre-formed at room temperature for 5 minutes. Then it was extruded and injected into any shape. The image shows that the bone cement material has good injectability and formability, and can effectively adapt to the treatment of defects of different sizes and shapes.

[0051] Figure 3 shows the mechanical properties of fully cured citrate-based bone cement prepared according to Examples 1 and 2. The bone cement was prepared into samples 12 mm high and 6 mm in diameter, and compressed to 50% or until fracture at a maximum force of 5 KN and a speed of 20 mm / min. The results show that toluene diisocyanate has higher molecular rigidity than 1,6-hexamethylene diisocyanate. Therefore, the strength of bone cement crosslinked with toluene diisocyanate is significantly improved, demonstrating the importance of the crosslinking agent in the preparation of citrate-based bone cement. This indicates that by changing the type of crosslinking agent, the compressive strength of the bone cement can be controlled as needed to meet the mechanical performance requirements of load-bearing bone sites.

[0052] Example 3 (1) 8 g of trimethyl citrate, 1 g of dioctyl adipate, 10 g of butanediol and 0.05 g of stannous octoate were added to a flask and reacted under vacuum at 160°C for 36 h. The reactants were precipitated with deionized water and obtained by freeze drying. (2) 1 g of the citrate prepolymer was weighed and added to a polytetrafluoroethylene (PTFE) mold. 0.3 g of toluene diisocyanate and 50 mg of calcium phosphate were added. The two components were stirred evenly and allowed to stand at room temperature for 5-15 minutes (the complete curing time in this example was 8.15 min) to obtain citrate bone cement.

[0053] Figure 4 shows the curing time of the citrate-based bone cement prepared according to the steps of Examples 2 and 3. The curing time was statistically analyzed by a rotational rheometer. The curing time of Example 2 was 8.44 minutes and the curing time of Example 3 was 8.15 minutes. This shows that the introduction of a small amount of additives does not affect the basic properties of the bone cement, and proves that the curing time of the bone cement meets the national standard requirement of 5 to 15 minutes, and is suitable for clinical use.

[0054] Figure 5 shows the curing temperature images of the citrate-based bone cement prepared according to the steps of Examples 2 and 3. The temperature was recorded every minute using an infrared camera and a curve was plotted. The highest temperature of the system in Example 2 during the entire curing process was 29.1℃, and the highest temperature of Example 3 was 27.5℃, proving that the introduction of a small amount of additives does not affect the basic properties of the bone cement, and that the cement has good safety during the curing process.

[0055] Figure 6 shows the compressive strength of the fully cured citrate-based bone cement prepared according to the steps of Example 3. The compressive strength test was the same as in Example 2. The results show that the compressive strength of the bone cement is 176.9 MPa, indicating that the introduction of additives can further improve the mechanical properties of the bone cement, proving that the material has controllability.

[0056] Figure 7 shows ALP staining images of the fully cured citrate-based bone cement prepared according to the steps of Examples 2 and 3. Citrate-based bone cement was prepared into sheets with a diameter of 8 mm and a height of 1 mm, sterilized, and then added to 48-well plates. 30,000 mouse bone marrow mesenchymal stem cells (BMSCs) were added to each well. After co-culturing for 24 h, the solution was replaced with an equal volume of 300 μL of osteogenic differentiation induction medium (purchased from OriCell, model MUXMX-90021) for 3 days of induction before staining observation. The results showed that the osteogenic properties of the citrate-based bone cement were significantly improved after the addition of the osteogenic additive, demonstrating that the biological properties of this citrate-based bone cement can be regulated on demand by introducing the additive.

[0057] Example 4 (1) 8 g of trimethyl citrate, 1 g of dioctyl adipate, 10 g of butanediol and 0.05 g of stannous octoate were added to a flask and reacted under vacuum at 160°C for 36 h. The reactants were precipitated with deionized water and obtained by freeze-drying. (2) 1 g of the citrate prepolymer was weighed and added to a polytetrafluoroethylene (PTFE) mold. 0.1 g of isophorone diisocyanate was added and the two components were stirred evenly. The mixture was allowed to stand at room temperature for 5-15 minutes (the complete curing time in this example was 10.5 min) to obtain citrate bone cement.

[0058] Example 5 (1) 8 g of trimethyl citrate, 1 g of dioctyl adipate, 10 g of butanediol and 0.05 g of stannous octoate were added to a flask and reacted under vacuum at 160°C for 36 h. The reactants were precipitated with deionized water and obtained by freeze drying. (2) 1 g of the citrate prepolymer was weighed and added to a polytetrafluoroethylene (PTFE) mold. 0.3 g of isophorone diisocyanate was added and the two components were stirred evenly. The mixture was allowed to stand at room temperature for 5-15 minutes (the complete curing time in this example was 10.1 min) to obtain citrate bone cement.

[0059] Example 6 (1) 8 g of trimethyl citrate, 1 g of dioctyl adipate, 10 g of butanediol and 0.05 g of stannous octoate were added to a flask and reacted under vacuum at 160°C for 36 h. The reactants were precipitated with deionized water and obtained by freeze-drying. (2) 1 g of the citrate prepolymer was weighed and added to a polytetrafluoroethylene (PTFE) mold. 0.5 g of isophorone diisocyanate was added and the two components were stirred evenly. The mixture was allowed to stand at room temperature for 5-15 minutes (the complete curing time in this example was 9.86 min) to obtain citrate bone cement.

[0060] Example 7 (1) 8 g of trimethyl citrate, 1 g of dioctyl adipate, 10 g of butanediol and 0.05 g of stannous octoate were added to a flask and reacted under vacuum at 160°C for 36 h. The reactants were precipitated with deionized water and obtained by freeze drying. (2) 1 g of the citrate prepolymer was weighed and added to a polytetrafluoroethylene (PTFE) mold. 0.7 g of isophorone diisocyanate was added and the two components were stirred evenly. The mixture was allowed to stand at room temperature for 5-15 minutes (the complete curing time in this example was 9.36 min) to obtain citrate bone cement.

[0061] Example 8 (1) 8 g of trimethyl citrate, 1 g of dioctyl adipate, 10 g of butanediol and 0.05 g of stannous octoate were added to a flask and reacted under vacuum at 160°C for 36 h. The reactants were precipitated with deionized water and obtained by freeze drying. (2) 1 g of the citrate prepolymer was weighed and added to a polytetrafluoroethylene (PTFE) mold. 1 g of isophorone diisocyanate was added and the two components were stirred evenly. The mixture was allowed to stand at room temperature for 5-15 minutes (the complete curing time in this example was 9.21 min) to obtain citrate bone cement.

[0062] Figure 8 shows the compressive strength of the fully cured citrate-based bone cement prepared according to the steps of Examples 4-8. The compressive strength test was the same as in Example 2. The results show that the strength of the prepared citrate-based bone cement increases with the increase of the amount of crosslinking agent introduced, proving that the material has controllability.

[0063] Figure 9 shows the surface modulus image of the fully cured citrate-based bone cement prepared according to the steps of Examples 5-8. The surface modulus of the bone cement was detected using a nanoindenter (KLA, G200X). The results show that the surface modulus of the prepared citrate-based bone cement also increases with the increase of the amount of crosslinking agent introduced, proving that the material is controllable and its mechanical properties meet the mechanical strength requirements of the load-bearing parts.

[0064] Example 9 (1) 8 g of trimethyl citrate, 1 g of dioctyl adipate, 9.5 g of butanediol, 0.5 g of N-methyldiethanolamine and 0.05 g of stannous octoate were added to a flask and reacted under vacuum at 160°C for 36 h. The reactants were precipitated with deionized water and obtained by freeze drying. (2) 1 g of the citrate prepolymer was weighed and added to a polytetrafluoroethylene (PTFE) mold. 0.5 g of isophorone diisocyanate was added and the two components were stirred evenly. The mixture was allowed to stand at room temperature for 5-15 minutes (10.5 min for complete curing in this example) to obtain citrate bone cement.

[0065] Example 10 (1) 8 g of trimethyl citrate, 1 g of dioctyl adipate, 9 g of butanediol, 1 g of N-methyldiethanolamine and 0.05 g of stannous octoate were added to a flask and reacted under vacuum at 160°C for 36 h. The reactants were precipitated with deionized water and obtained by freeze-drying. (2) 1 g of citrate prepolymer was weighed and added to a polytetrafluoroethylene (PTFE) mold. 0.5 g of isophorone diisocyanate was added and the two components were stirred evenly. The mixture was allowed to stand at room temperature for 5-15 minutes (the complete curing time in this example was 11.6 min) to obtain citrate bone cement.

[0066] Figure 10 shows the degradation rate of fully cured citrate-based bone cement prepared according to Examples 6, 9, and 10. Citrate-based bone cement was formed into sheets with a diameter of 8 mm and a height of 1 mm, placed in 5 mL of PBS (pH=7.40), and placed in a shaker at 37°C and 100 rpm. The solution was changed every 3 days, and the mass change was measured after 5 days and 15 days. The degradation amount was calculated as: original mass of citrate-based bone cement - mass of citrate-based bone cement at 5 days / mass of citrate-based bone cement at 15 days. The results show that the prepared citrate-based bone cement is degradable, and increasing the amount of N-methyldiethanolamine introduced can effectively improve the degradation rate of citrate-based bone cement. This demonstrates that changing the diol structure of the prepolymer can effectively improve the degradation rate of citrate-based bone cement, and that the material is designable and can better meet clinical needs.

[0067] Figure 11 shows images of the citric acid release from the fully cured citrate-based bone cement prepared according to Examples 6, 9, and 10. The left image shows the citric acid content released into the supernatant, and the right image shows the citric acid content in the cells after co-incubation with cells. Citrate-based bone cement was fabricated into sheets with a diameter of 8 mm and a height of 1 mm. After sterilization, the sheets were added to 48-well plates, and 500 μL of culture medium containing 30,000 mouse bone marrow mesenchymal stem cells (BMSCs) was added to each well. After co-culturing for 24 h, the supernatant and cells on the material surface were collected, and the citric acid content in the supernatant and cells was measured using a kit. The results showed that the material could degrade and release citric acid, and the material that degraded faster released a correspondingly higher amount of citric acid, resulting in a corresponding increase in the intracellular citric acid content.

[0068] Example 11 (1) 8 g of trimethyl citrate, 1 g of dioctyl adipate, 10 g of butanediol and 0.05 g of stannous octoate were added to a flask and reacted under vacuum at 160°C for 36 h. The reactants were precipitated with deionized water and obtained by freeze-drying. (2) 1 g of citrate prepolymer was weighed and added to a polytetrafluoroethylene (PTFE) mold. 1 g of isophorone diisocyanate and 10 mg of inorganic ceramic were added. The two components were stirred evenly and allowed to stand at room temperature for 5-15 minutes (the complete curing time in this example was 8.3 min) to obtain citrate bone cement that promotes angiogenesis.

[0069] Figure 12 is a cross-sectional SEM image of the fully cured citrate-based bone cement prepared according to Example 11. The spherical structures marked by arrows in the figure are encapsulated inorganic ceramic particles. The convex and concave structures indicate that the inorganic ceramics were successfully and uniformly filled in the citrate-based bone cement.

[0070] Figure 13 shows the angiogenic properties of the fully cured citrate-based bone cement prepared according to Examples 8 and 11. Citrate-based bone cement was formed into sheets with a diameter of 8 mm and a height of 1 mm. After sterilization, the sheets were immersed in 1 mL of commercially available ECM culture medium in 48-well plates and incubated at 37°C for 24 h to obtain the extract. In 24-well plates, matrix gel (purchased from Corning, model 356234) was layered, and 200,000 human umbilical vein endothelial cells (HUVECs) were added to each well. The corresponding extracts were added, and the plates were cultured for 4 h before observing their angiogenic properties. The results show that the citrate-based bone cement exhibited better angiogenic properties after the addition of angiogenic inorganic ceramics. The vascular network formed in Example 11 was significantly better than that in Example 8 without inorganic ceramics. This demonstrates that the bone cement material can be controlled as needed to prepare angiogenic bone cement to meet the requirements of bone defect sites.

[0071] Figure 14 shows cell images on the surface of fully cured citrate-based bone cement prepared according to Examples 8, 9, and 11. Citrate-based bone cement was prepared into sheets with a diameter of 8 mm and a height of 1 mm, sterilized, and then added to 48-well plates. 500 μL of culture medium containing 30,000 mouse bone marrow mesenchymal stem cells (BMSCs) was added to each well. After co-culturing for 24 h, the cells were fixed with tissue fixation solution and stained for observation. The results showed that the cells were evenly spread on the surface of the citrate-based bone cement material and grew well, demonstrating that the bone cement material has good biocompatibility.

[0072] Figure 15 shows the blood compatibility images of the fully cured citrate-based bone cement prepared according to Examples 8, 9, and 11. 1 mL of fresh rat blood was added to 15 mL of physiological saline and pumped at 2000 rpm for [time missing]. -1 Centrifuge for 15 min, wash, discard supernatant, add fresh physiological saline, and repeat washing three times. Discard supernatant, dilute red blood cells with physiological saline to prepare a 2% hemoglobin solution, and weigh 2 mg of Triton X-100 into 2 mL of 2% hemoglobin solution to prepare a 1% m / v Triton X-100 solution. Prepare citrate-based bone cement sheets with a diameter of 8 mm and a height of 1 mm and place them in a 48-well plate. Add 500 μL of 2% hemoglobin solution to each well. Add 2% hemoglobin solution as a negative control and 1% Triton X-100 solution as a positive control. Incubate at 37°C for 3 h. Then transfer the liquid in the plate to centrifuge tubes and centrifuge. Collect the supernatant and centrifuge. 545 nm The absorbance value was measured at the sample site, and the hemolysis rate of the sample was calculated.

[0073] The formula for calculating the hemolysis rate is as follows: Hemolysis rate (%) = (A 样 -A 阴 ) / (A 阳 -A 阴 )×100%; where A 样 This refers to the absorbance value of each sample, A. 阴 This refers to the absorbance value of a 2% hemoglobin solution, A. 阳 This refers to the absorbance value of 1% Triton X-100.

[0074] As shown in Figure 15, the hemolysis rate of all material groups was less than 5%, indicating that the citrate-based bone cement material has good blood compatibility.

[0075] Example 12 (1) 6 g of tributyl citrate, 3 g of diethyl tartrate, 10 g of octanediol and 0.05 g of stannous octoate were added to a flask and reacted under vacuum at 160°C for 36 h. The reactants were precipitated with deionized water and obtained by freeze-drying. (2) 1 g of citrate prepolymer was weighed and added to a PTFE mold. 0.5 g of isophorone diisocyanate and 10 mg of vancomycin were added. The two components were stirred evenly and allowed to stand at room temperature for 5-15 minutes (the complete curing time in this example was 6.05 min) to obtain antibacterial citrate bone cement.

[0076] Figure 16 shows the inhibition zone of fully cured citrate-based bone cement prepared according to Examples 1 and 12. Staphylococcus aureus was added to LB broth and incubated overnight at 37°C. Bacterial density was determined using NanoDrop One (Thermo, USA), and the bacterial suspension concentration was then diluted to 10⁻⁶. 5 CFU mL -1 50 μL of diluted bacterial suspension was evenly spread onto an LB agar plate. The UV-sterilized sample was placed face down on the LB agar plate coated with bacterial suspension, and then incubated at 37°C for 24 h. The presence of an inhibition zone around the sample was observed. The image shows a clear inhibition zone around the antibiotic-treated bone cement, proving that the added antibiotic can be successfully released from the bone cement to kill bacteria and has good potential for preventing bacterial infection in vivo.

[0077] Example 13 (1) 6 g of tributyl citrate, 3 g of diethyl tartrate, 10 g of octanediol, 1 g of catalol and 0.05 g of stannous octoate were added to a flask and reacted under vacuum at 160°C for 36 h. The reactants were precipitated with deionized water and obtained by freeze drying to form liquid citrate prepolymer. (2) 1 g of citrate prepolymer was weighed and added to a PTFE mold, and 0.5 g of isophorone diisocyanate was added. The two components were stirred evenly and allowed to stand at room temperature for 5-15 minutes (the complete curing time in this example was 7.12 min) to obtain anti-inflammatory citrate bone cement.

[0078] Figure 17 shows stained images of fully cured citrate-based bone cement prepared according to Examples 6 and 13, co-cultured with macrophages. Citrate-based bone cement was prepared into sheets with a diameter of 8 mm and a height of 1 mm, sterilized, and added to 48-well plates. 200 μL of culture medium containing 30,000 mouse mononuclear macrophages (RAW264.7) was added to each well. After co-culturing for 24 h, the cells were fixed with tissue fixative (Solepro, model P1110) and stained for observation. The results showed that catalpol has anti-inflammatory and antioxidant properties, therefore, the citrate-based bone cement with catalpol incorporation possesses anti-inflammatory properties. Macrophages on the surface of Example 13 were polarized towards M2, while those on the surface of Example 6 were polarized towards M1, demonstrating that anti-inflammatory bone cement can be prepared by adjusting the molecular structure, thereby promoting the repair of bone defects.

[0079] Figure 18 shows the RNA sequencing data of the fully cured citrate-based bone cement prepared according to Example 13. The left image is a volcano plot, and the right image shows differentially regulated genes. With the pure cell group as the control group, the sequencing results showed that the modified anti-inflammatory citrate bone cement had a relatively high osteogenic differentiation capacity, maintained the proliferation and differentiation balance required for osteogenic formation, and significantly reduced inflammatory factors such as Tnf, ll1b, ll6, and Nlrp3, indicating that this bone cement material can effectively inhibit the inflammatory response and promote damage repair.

[0080] Figure 19 shows the in vivo curing of the fully cured citrate bone cement prepared according to Example 13. A 2 mm wide and 1 mm deep hole was drilled in the femur of a mouse. The bone cement prepolymer was mixed and loaded into a 1 mL syringe. After pre-forming at room temperature for 5 minutes, it was injected into the defect site. It cured at the defect site in approximately 1 minute, allowing for suturing. A CT scan was performed 3 days later. The results showed that the injected bone cement remained stably in the defect site without any spillage, indicating that the bone cement material also has good curing properties in vivo and can effectively treat defects of different shapes and sizes.

[0081] Comparative Example 1 (1) 7 g of trimethyl citrate, 3 g of dioctyl adipate, 6 g of octyl glycol and 0.05 g of stannous octoate were added to a flask and reacted under vacuum at 160°C for 48 h. The reactants were precipitated with deionized water and obtained by freeze-drying. (2) 1 g of the citrate prepolymer was weighed and added to a PTFE mold, and 0.3 g of hexamethylene diisocyanate was added. The two components were stirred evenly and allowed to stand at room temperature for 5-50 minutes (the complete curing time in this example was 45.26 min) to obtain citrate bone cement.

[0082] Comparative Example 2 (1) 7 g of trimethyl citrate, 3 g of dioctyl adipate, 5 g of octyl glycol, 1 g of isosorbide and 0.05 g of stannous octoate were added to a flask and reacted under vacuum at 160°C for 48 h. The reactants were precipitated with deionized water and obtained by freeze-drying. (2) 1 g of citrate prepolymer was weighed and added to a PTFE mold, and 0.3 g of hexamethylene diisocyanate was added. The two components were stirred evenly and allowed to stand at room temperature for 5-50 minutes (the complete curing time in this example was 40.76 min) to obtain citrate bone cement.

[0083] Figure 20 shows the curing time of citrate bone cement prepared according to the preparation methods of Comparative Example 1 and Comparative Example 2. The left figure shows the curing time of citrate bone cement prepared according to the preparation method of Comparative Example 1, and the right figure shows the curing time of citrate bone cement prepared according to the preparation method of Comparative Example 2. The results show that when the amount of polyol is less than the total amount of citrate polyester and reinforcing polyester, the curing time of citrate bone cement will be greatly prolonged, resulting in prolonged operation time, which is not conducive to clinical use.

[0084] Comparative Example 3 (1) 6 g of tributyl citrate, 3 g of diethyl tartrate, 10 g of octanediol and 0.05 g of stannous octoate were added to a flask and reacted under vacuum at 160°C for 36 h. The reactants were precipitated with deionized water and obtained by freeze-drying. (2) 1 g of the citrate prepolymer was weighed and added to a PTFE mold, and 0.5 g of lysine diisocyanate was added. The two components were stirred evenly and allowed to stand at room temperature for 5-15 minutes (the complete curing time in this example was 14.3 min) to obtain citrate bone cement.

[0085] Figure 21 is a picture of the fully cured citrate bone cement prepared according to the preparation method of Comparative Example 3 after pressure test. The compressive strength test is the same as in Example 2. The results show that when the crosslinking agent is not properly selected, the compressive strength of the prepared bone cement is insufficient, and it will crack after being compressed, making it unsuitable for repairing bone defects in load-bearing parts.

[0086] Comparative Example 4 (1) 6 g of tributyl citrate, 3 g of diethyl tartrate, 10 g of bis(2-hydroxyethyl) disulfide and 0.05 g of stannous octoate were added to a flask and reacted under vacuum at 160°C for 36 h. The reactants were precipitated with deionized water and obtained by freeze-drying. (2) 1 g of the citrate prepolymer was weighed and added to a PTFE mold, and 0.5 g of hexamethylene diisocyanate was added. The two components were stirred evenly and allowed to stand at room temperature for 5-15 minutes (the complete curing time in this example was 2.35 min) to obtain citrate bone cement.

[0087] Figure 22 shows the curing temperature of the citrate bone cement prepared according to the preparation method of Comparative Example 4. The results show that when the reaction rate of the selected diol is too fast, the bone cement will rapidly release heat during the curing process, and the temperature will reach 93.3℃. The high temperature is likely to damage the surrounding cells and is not conducive to defect repair. At the same time, the large amount of heat generated due to the rapid reaction rate results in a short curing time, which is also not conducive to use.

[0088] Comparative Example 5 (1) 9 g of trimethyl citrate, 10 g of butanediol and 0.05 g of stannous octoate were added to a flask and reacted under vacuum at 160°C for 36 h. The reactants were precipitated with deionized water and obtained by freeze drying. (2) 1 g of the citrate prepolymer was weighed and added to a polytetrafluoroethylene (PTFE) mold. 0.3 g of isophorone diisocyanate was added and the two components were stirred evenly. The mixture was allowed to stand at room temperature for 5-15 minutes (the complete curing time in this example was 9.8 min) to obtain citrate bone cement.

[0089] Figure 23 shows the image after pressure testing of the fully cured citrate ester bone cement prepared according to the preparation method of Comparative Example 5. The compressive strength test was the same as in Example 2. The results show that without the addition of reinforcing polyester, the bone cement prepared by using only citrate ester for prepolymer preparation has a compressive strength of only about 10 MPa, which is more elastic and not suitable for the mechanical performance requirements of load-bearing bone parts. However, the strength of the bone cement prepared by introducing reinforcing polyester in Example 5 is much higher than the 70 MPa strength of PMMA bone cement. This proves the importance of reinforcing polyester in the preparation of citrate ester-based bone cement and shows that the mechanical properties of bone cement can be controlled as needed through the design of the prepolymer molecular structure to meet the mechanical performance requirements of load-bearing bone parts.

[0090] Comparative Example 6 (1) 8 g of trimethyl citrate, 1 g of dioctyl adipate, 5 g of butanediol, 5 g of N-methyldiethanolamine and 0.05 g of stannous octoate were added to a flask and reacted under vacuum at 160°C for 36 h. The reactants were precipitated with deionized water and obtained by freeze-drying. (2) 1 g of citrate prepolymer was weighed and added to a polytetrafluoroethylene (PTFE) mold. 0.5 g of isophorone diisocyanate was added and the two components were stirred evenly. The mixture was allowed to stand at room temperature for 5-50 minutes (the complete curing time in this example was 48.6 min) to obtain citrate bone cement.

[0091] Figure 24 is a physical image of the fully cured citrate prepolymer prepared according to the preparation methods of Comparative Example 6 and Example 10. The left image is Comparative Example 6 and the right image is Example 10. The results show that if too much N-methyldiethanolamine is introduced, the viscosity of the prepolymer will be too high and it will lose its fluidity, which is not conducive to injection.

[0092] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing bone cement, characterized in that, include: Components A, B, and C are reacted to form a citrate-based prepolymer. Component A is a citrate polyester containing at least two reactive ester functional groups on its terminal group or side chain; Component B is a non-citrate polyester containing at least two reactive functional groups on its terminal group or side chain, wherein the functional groups are the same or different; the functional groups are selected from at least one of ester group, hydroxyl group, amino group, mercapto group, epoxy group, carboxyl group, acid anhydride, and organic substituent; Component C is a polyol; the reaction includes transesterification reaction, or a combination of transesterification reaction and at least one of polycondensation reaction and substitution reaction; bone cement is prepared by taking citrate ester prepolymer and isocyanate, and crosslinking reaction of urethane bond or amide bond is carried out.

2. The method for preparing bone cement according to claim 1, characterized in that, The citric acid polyester is at least one of dimethyl citrate, trimethyl citrate, diethyl citrate, triethyl citrate, dipropyl citrate, or tributyl citrate; and / or, component B is a reinforcing polyester selected from at least one of phthalate, dioctyl adipate, dioctyl sebacate, isooctyl mercaptoacetate, methyl 3-aminoacrylate, pentaerythritol ester, or diethyl tartrate; and / or, the polyol is at least one of butanediol, octyl glycol, bis(4-hydroxyphenyl) disulfide, resveratrol, bis(2-hydroxyethyl) disulfide, isosorbide, polyethylene glycol, xylitol, citric acid alcohol, or N-methyldiethanolamine.

3. The method for preparing bone cement according to any one of claims 1-2, characterized in that, The step of forming the citrate-based prepolymer also includes a catalyst, wherein the catalyst is at least one of stannous octoate, dibutyltin dilaurate, or zinc chloride.

4. The method for preparing bone cement according to any one of claims 1-3, characterized in that, The isocyanate is selected from at least one of isophorone diisocyanate, 1,6-hexamethylene diisocyanate, toluene diisocyanate, naphthalene diisocyanate, 1,4-cyclohexyl diisocyanate, or 4,4'-diphenylmethane diisocyanate.

5. The method for preparing bone cement according to any one of claims 1-4, characterized in that, In preparing bone cement, additives are also included, wherein the additives are selected from bone-promoting additives, contrast agents, or antibacterial agents; the bone-promoting additives are selected from at least one of hydroxyapatite, magnesium hydroxide, calcium phosphate, inorganic ceramics, plexiglass, alendronate, or clay; the contrast agents are selected from at least one of zirconium oxide, barium sulfate, strontium polyphosphate, strontium salts, graphene-iodohydrin, or tungsten / bismuth oxide; and the antibacterial agents are selected from at least one of antibiotics, antimicrobial peptides, inorganic metal ions, or quaternary ammonium salts.

6. The method for preparing bone cement according to any one of claims 3-5, characterized in that, The product comprises the following raw materials, by weight: 1-15 parts by weight of citric acid polyester, 1-15 parts by weight of component B, 2-30 parts by weight of polyol, and 0.001-2 parts by weight of catalyst; wherein the weight of polyol is greater than or equal to the sum of the weight of citric acid polyester and reinforcing polyester; wherein isocyanate is 0.1-100 parts by weight, and osteopromoting additive is 0-10 parts by weight.

7. The method for preparing bone cement according to any one of claims 1-6, characterized in that, In the step of forming the citrate ester prepolymer, the reaction conditions are a temperature of 140~180℃ and a reaction time of 24~48 h.

8. The method for preparing bone cement according to any one of claims 1-7, characterized in that, The conditions for the crosslinking reaction of the urethane or amide bonds are: room temperature, curing time of 0-20 min, and reaction time > 0 min, and injectability before complete curing; the exothermic temperature during curing is 0-45℃, and ≠ 0℃.

9. A bone cement prepared by the bone cement preparation method according to any one of claims 1-8; optionally, the compressive strength of the bone cement is 0~200 MPa and the compressive modulus is 0~2 GPa.

10. The use of the bone cement as described in claim 9 in the preparation of orthopedic, neurosurgical or craniofacial reconstructive materials.

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