Bone cement material as well as preparation method and application thereof

Abstract

The invention discloses a bone cement material as well as a preparation method and application thereof, bone morphogenetic protein-2 is doped in a bone cement raw material containing the components of PEGS-OH and CPC, and the bone cement material is prepared under the action of a cross-linking agent, under the condition that a very small amount of bone morphogenetic protein-2 is added into the bone cement material, the bone cement material with good slow release stability, high osteogenic activity and the like can be obtained.

Description

Technical Field

[0001] The present invention relates to a bone cement material, a preparation method and an application thereof, and in particular to a bone cement material, a preparation method and an application thereof. Background Art

[0002] The increasing incidence of bone defects caused by illness, accidental injury, and natural disasters has not only created a huge market demand for bone repair materials, but also placed increasingly stringent demands on existing materials in terms of surgical operability, mechanical properties, and bioactivity. Polymethyl methacrylate (PMMA) bone cement is the most widely used. However, PMMA bone cement is bioinert, non-degradable, and the high temperatures (approximately 70°C) generated during its reaction significantly impair its tissue repair efficacy, rendering it limited to a filler and unable to guide bone regeneration. Consequently, the more biocompatible calcium phosphate cement (CPC) has been developed and used to immobilize and deliver bone morphogenetic protein-2 (rhBMP-2) to enhance its osteogenic activity. However, this binding mechanism is solely physical adsorption, resulting in a burst of BMP-2 release. This leads to excessive protein concentrations early in the repair phase and insufficient concentrations later in the repair phase, ultimately resulting in the formation of deformed bone spurs and incomplete repair.

[0003] Therefore, there is an urgent need for an injectable bone cement material with better osteogenesis in the market. Summary of the Invention

[0004] Purpose of the invention: In order to solve the problem in the prior art that rhBMP-2 has a high initial instantaneous concentration and uneven release, resulting in poor osteogenesis, the purpose of the present invention is to provide an injectable bone cement material that can achieve a stable sustained release and high osteogenesis activity by adding a very small amount of rhBMP-2, as well as a preparation method and use.

[0005] Previous studies by the present invention's research team have shown that the introduction of PEGylated polysebacic acid (PEGS) can effectively improve CPC's brittleness and poor anti-collapse properties, but the factors influencing its osteogenic properties have not been thoroughly studied. This time, the present invention's team focused on the effects of bone morphogenetic protein-2 (rhBMP-2) on the osteogenic properties of bone cement materials.

[0006] Technical solution: On one hand, the present invention provides a bone cement material, wherein bone morphogenetic protein-2 is doped into a bone cement raw material composed of polyester polymer PEGS-OH and calcium phosphate salt CPC, and the bone cement material is prepared under the action of a cross-linking agent.

[0007] Furthermore, the mass ratio of the polyester polymer PEGS-OH, calcium phosphate CPC and bone morphogenetic protein-2 is 2 mg:8 mg:(1 ug-5 ug).

[0008] Furthermore, the mass ratio of the polyester polymer PEGS-OH, calcium phosphate CPC and bone morphogenetic protein-2 is 2mg:8mg:5ug.

[0009] The second aspect of the present invention provides a method for preparing the bone cement material, comprising taking polyester polymer PEGS-OH, calcium phosphate CPC and a cross-linking agent, mixing them evenly, adding bone morphogenetic protein-2, and stirring to obtain bone cement slurry, i.e., bone cement material.

[0010] Furthermore, the cross-linking agent is first mixed evenly with the bone morphogenetic protein-2.

[0011] Furthermore, the cross-linking agent is LDI cross-linking agent.

[0012] The third aspect of the present invention provides uses of the bone cement material, including any one of the following: (i) preparing tissue defect filling materials; (ii) preparing materials to guide tissue regeneration; (iii) preparation of medical cosmetic materials; (iv) preparing 3D printing matrix materials; (v) Serves as a loading matrix or carrier for growth factors or drugs.

[0013] The principles of the present invention are as follows: The sustained release of BMP-2 by organic phases ensures its long-term release and reduces the instantaneous concentration of BMP-2, preventing short-term high concentrations that could lead to side effects such as bone spurs, enhanced osteoclastogenesis, and suboptimal osteogenesis. BMP-2 and bone cement are closely linked. BMP-2 induces the differentiation of bone marrow mesenchymal stem cells into osteoblasts, significantly enhancing the osteogenic activity of PEGS / CPC injectable bone cement. However, bone regeneration requires more than osteoblasts; calcium and phosphorus, essential components of bone tissue, are also required. Therefore, CPC in bone cement provides a stable supply of calcium and phosphate, providing the raw materials for bone regeneration. Furthermore, PEGS is composed of sebacic acid, glycerol, and polyethylene glycol. Studies have shown that sebacic acid participates in the tricarboxylic acid cycle, providing cells with adenosine triphosphate (ATP), thereby providing energy and further promoting osteogenesis. As a biodegradable and resorbable material, PEGS / CPC injectable bone cement cannot persist permanently in the body like PMMA. Therefore, it is very important to enhance its osteogenic activity to induce bone regeneration.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. A small amount of rhBMP-2 can achieve excellent osteogenic activity. Adding a small amount of rhBMP-2 can achieve excellent sustained-release properties and osteogenic activity while maintaining unchanged appearance and fluidity. Alizarin red mineralization deposition (Figure C) and alkaline phosphatase staining (Figure D) of rhBMP-2-loaded bone cement demonstrate that the osteogenic activity of the material gradually increases with increasing rhBMP-2 protein content. This is due to the fact that rhBMP-2, as a functional protein, promotes the differentiation of bone marrow mesenchymal stem cells into osteoblasts.

[0015] 2. Good sustained-release effect. Small release amount: The organic phase PEGS in PEGS / CPC contains a large number of ester and ether bonds, which can form a large number of hydrogen bonds with the rhBMP-2 protein, thereby enhancing the immobilization rate of rhBMP-2 and achieving sustained release of rhBMP-2, which is beneficial to osteogenesis. Especially in the initial release period, the release amount is small, avoiding the initial burst release that may lead to suboptimal osteogenesis.

[0016] 3. Reduced burst release. The presence of PEGS in the bone cement of this invention forms numerous hydrogen bonds with rhBMP-2, resulting in sustained release. The amount of rhBMP-2 released over two weeks is only 50 ng, or 5% of the total dose, and the release curve subsequently flattens. Conventional pure CPC releases a significant burst, particularly within the first day, but releases approximately 60% within two weeks.

[0017] 4. The addition of different amounts of rhBMP-2 had no significant effect on the cytotoxicity of the material, and the cell compatibility of each group was higher than 70% required by the national standard "GB / T 16886.5".

[0018] 5. Appearance remains unchanged: The amount of rhBMP-2 added is very small compared to bone cement, so it does not significantly interfere with the performance of bone cement. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The appearance of PEGS / CPC bone cement when doped with different contents of rhBMP-2: A: 400 mg bone cement doped with 1 ug rhBMP-2; B: 400 mg bone cement doped with 3 ug rhBMP-2; C: 400 mg bone cement doped with 5 ug rhBMP-2. Figure 2 is the cytotoxicity test result; Figure 3 The release curve of rhBMP-2 in bone cement within 2 weeks was shown. 5 μg of rhBMP-2 was added; Figure 4 The release curve of rhBMP-2 in bone cement within 2 weeks was shown. 3 μg of rhBMP-2 was added; Figure 5 The release curve of rhBMP-2 in bone cement within 2 weeks was shown. 1 μg of rhBMP-2 was added; Figure 6 The release curve of rhBMP-2 in bone cement within 2 weeks was 8ug rhBMP-2 added; Figure 7 The release curve of rhBMP-2 in bone cement within 2 weeks was shown. 10 μg of rhBMP-2 was added; Figure 8 The release curve of rhBMP-2 in bone cement over 2 weeks: ordinary pure CPC with 5 μg rhBMP-2 added (normal control); Figure 9 Quantitative analysis of Alizarin red mineralization deposition of rhBMP-2 loaded in bone cement; Figure 10 For quantitative analysis of alkaline phosphatase staining; Figure 11 This is a photo of alizarin red mineralized deposits; Figure 12 This is a photo of alkaline phosphatase staining. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be further described below.

[0021] Example 1 Preparation of PEGS / CPC / rhBMP-2 The mass ratio of polyester polymer PEGS-OH to calcium phosphate salt CPC is fixed at 2:8. This ratio creates a bone cement that is between liquid and solid, making it suitable for injection. The following study investigated the effects of adding different amounts of rhBMP-2 on the osteogenic capacity and sustained-release properties of the bone cement.

[0022] Weigh 80mg PEGS-OH, 320mg CPC, and 26.4uL LDI in triplicate, mix well, and add 1ug, 3ug, and 5ug of rhBMP-2, respectively, to form bone cement slurry ( Figure 1 ).Depend on Figure 1 It can be seen that after adding rhBMP-2, bone cement still has good fluidity. Figure 1 As can be seen, the bone cement has a viscous toothpaste-like consistency. Since the amount of BMP-2 added is extremely small, its addition has no significant effect on the state of the bone cement, resulting in good viscosity and fluidity. However, as the amount of rhBMP-2 increases, such as when 8 μg of rhBMP-2 is added, the fluidity and viscosity deteriorate, making it uninjectable and unusable as a bone material.

[0023] Example 2 Characterization of Cytotoxicity of Bone Cement of the Present Invention In order to evaluate the cytocompatibility of bone cement, this study used a culture method of mouse fibroblasts (L929) added with bone cement extract, and investigated the cytotoxicity of the cells and the extract after culturing for 24 hours.

[0024] First, prepare rhBMP-2-containing bone cement in a clean hood: weigh 80 mg of PEGS-OH, 320 mg of CPC, and 1 μg, 2 μg, and 3 μg of rhBMP-2 lyophilized powder, sterilized by UV. The crosslinker, LDI, was sterilized by filtration before use. RhBMP-2 was thoroughly mixed with 26.4 μL of LDI and then blended with PEGS-OH and CPC to form a bone cement slurry. A blank control group was prepared without rhBMP-2, resulting in four bone cements with varying rhBMP-2 contents (B0, B1, B2, and B3). Allow to fully cure at 37°C for 24 hours. To prepare the extract, add 2 mL of DMEM medium to the bone cement at a ratio of 200 mg of bone cement to 1 mL of medium. Simultaneously, L929 cells were plated in a 96-well plate (1 × 10 4 Six replicates were prepared for each group and placed in a cell culture incubator maintained at a constant temperature and humidity of 95% air / 5% carbon dioxide. After 24 hours, the culture medium was discarded, and the extract (100 μL / well) was added. The cells were then cultured in the cell culture incubator for another 24 hours. The samples were then subjected to an MTT assay to assess the cytotoxicity of the bone cement, in accordance with the national standard GB / T 16886.5-2017. Figure 2 The cytotoxicity results showed that bone cement (group B0) itself had certain toxicity to cells, and the addition of rhBMP-2 (B1, B2, and B3) had no significant effect on cell toxicity, all meeting the national standard requirements.

[0025] Example 3 Characterization of the release kinetics of rhBMP-2 in the bone cement of the present invention 1. Add 5ug rhBMP-2 The cumulative release curve of rhBMP-2 in bone cement was detected using an ELISA kit. 80 mg PEGS-OH, 320 mg CPC, 26.4 μL LDI, and 5 μg rhBMP-2 freeze-dried powder were weighed. LDI was first mixed thoroughly with rhBMP-2, and then mixed with PEGS-OH and CPC. The mixture was stirred thoroughly to form a bone cement slurry. The bone cement was placed in a 6 mm diameter, 6 mm deep, cylindrical polytetrafluoroethylene mold. After fully curing at 37°C, the mold was removed. The bone cement was submerged in 2 mL of PBS solution. At pre-determined time points, 2 mL of sustained-release solution was removed and then added to continue the sustained-release process. The samples of sustained-release solution removed at each time point were numbered and stored frozen at -20°C. The rhBMP-2 concentration in the sustained-release solution was determined using an rhBMP-2 ELISA kit according to the kit instructions. The OD value at 450 nm was measured using a SPECTRAmax 384 microplate reader. The curve equation was fitted to the standard curve, and the amount of rhBMP-2 released at each time point was calculated.

[0026] Figure 3 The cumulative release curve of rhBMP-2 within 14 days is shown. In the first 7.5 hours, there is a small burst release of rhBMP-2, and then the release rate slows down. The overall release is relatively stable and can be released continuously, which is beneficial to osteogenesis and activity improvement.

[0027] 2. Add 3ug rhBMP-2 The method is the same as 1, except that 3ug of rhBMP-2 freeze-dried powder is added.

[0028] Figure 4 The cumulative release curve of rhBMP-2 within 14 days shows that it is released rapidly in the first 11 hours, and then the release rate slows down. The overall release is relatively stable and can be released continuously, which is beneficial to the improvement of osteogenesis and activity.

[0029] 3. Add 1ug rhBMP-2 The method is the same as 1, except that 1 μg of rhBMP-2 freeze-dried powder is added.

[0030] Figure 5 The cumulative release curve of rhBMP-2 within 14 days is shown. In the first 9 hours, rhBMP-2 is released rapidly, and then the release rate slows down. The overall release is relatively stable and can be released continuously, which is beneficial to the improvement of osteogenesis and activity.

[0031] 4. Add 8ug rhBMP-2 The method is the same as 1, except that 8ug of rhBMP-2 freeze-dried powder is added.

[0032] Figure 6The cumulative release curve of rhBMP-2 over 14 days shows a continuous and rapid release of rhBMP-2, reaching 2.5 μg within two weeks. Experiments have shown that the amount of rhBMP-2 added cannot be increased indefinitely. As the amount of rhBMP-2 added increases, the amount of rhBMP-2 released increases, the release rate accelerates, and the initial burst release is significant, which is not conducive to osteogenesis.

[0033] 5. Add 10ug rhBMP-2 The method is the same as 1, except that 10 μg of rhBMP-2 freeze-dried powder is added.

[0034] Figure 7 The cumulative release curve of rhBMP-2 over 14 days shows a continuous and rapid release of rhBMP-2, reaching 3 μg within 2 weeks. Experiments have shown that the amount of rhBMP-2 added cannot be increased indefinitely. As the amount of rhBMP-2 added increases, the amount of rhBMP-2 released increases, the release rate accelerates, and the initial burst release is significant, which is not conducive to osteogenesis.

[0035] also, Depend on Figure 3 、 Figure 4 、 Figure 5 It can be seen that the release amount of the bone cement prepared by the present invention is small within 2 weeks, with a small burst release in the first day, and then the release is relatively stable, which reduces the early BMP-2 protein concentration, ensures the stability of the later concentration, improves osteogenesis, and achieves a better repair effect.

[0036] Depend on Figure 6 、 Figure 7 Comparison shows that with increasing rhBMP-2 dosage, the initial burst release increases, and the later release rate increases, reaching over 2.5 μg within 2 weeks. This leads to uneven release of BMP-2 protein concentration, affecting osteogenesis and repair, causing "ectopic osteogenesis" and related "soft tissue inflammatory response." It can be seen that the more rhBMP-2 added, the better. The amount of rhBMP-2 added has a significant impact on the osteogenesis effect.

[0037] Depend on Figure 3 and Figure 8 By comparison, the release amount of the bone cement prepared by the present invention within 2 weeks is far less than that of ordinary pure CPC bone cement, and the early burst release amount is greatly reduced compared with ordinary pure CPC bone cement, indicating that the protein concentration of the osteogenic material prepared by the present invention will not be too high in the early stage of bone repair, ensuring the concentration in the later stage, which is beneficial to bone survival and repair.

[0038] Example 4 Characterization of the osteoinductive activity of the bone cement of the present invention ALP activity characterization: ALP kit was used to detect the osteogenic induction activity of PEGS-OH / CPC / rhBMP-2. S ) were co-cultured with bone cement in 24-well plates for 7 days and the ALP activity of the samples was detected. The experiment was set up in 6 groups, with 3 parallel samples in each group. Each sample weighed 1-2 mg. The Blank group did not add bone cement. Groups B0, B1, B2, B3, and B4 respectively added rhBMP-2 0ug, 1ug, 2ug, 3ug, and 4ug to the bone cement. The samples were first irradiated with UV light and then incubated with BMSCs. S Co-culture was performed on days 1, 3, and 5 of co-culture. Cells were replaced with osteogenic induction medium prepared in the OriCell Rat Bone Marrow Mesenchymal Stem Cell Osteogenic Differentiation Kit, and the cells were wrapped in aluminum foil to protect from light. Qualitative analysis of ALP was performed on day 7: 24-well plates were washed three times with PBS, fixed with 200 μL / well of 4% paraformaldehyde for 20 minutes, then washed three times with PBS, added with 200 μL / well of BCIP / NBT staining solution, and incubated at room temperature in the dark for 30 minutes before photographing. Quantitative analysis of ALP was performed: 100 μL / well of lysis buffer NP-40 was added, and cells were lysed at 37°C for 90 minutes. A 96-well plate was prepared, and samples, diluents, and color development solution were added. The plates were then incubated at 37°C in the dark for 30 minutes. Then, 100 μL of reaction stop solution was added to each well, and the OD at 405 nm was measured. Figure 10 The quantitative results of ALP show that as the content of rhBMP-2 gradually increases, rhBMP-2 has a significant effect on promoting BMSC growth. S However, as the amount increases (such as above 5ug), the activity tends to slow down, and it will lead to cystic bone formation and significant soft tissue swelling. Therefore, the dosage of rhBMP-2 should not be added arbitrarily. Figure 12 The qualitative results of ALP indicate that the inorganic phase CPC in bone cement provides a large amount of calcium, phosphorus and other elements, which itself has the ability to promote osteogenic differentiation (group B0). As the content of rhBMP-2 increases, the osteogenic ability of bone cement is greatly improved, but as the amount increases (such as above 5ug), the activity tends to be slow, and it will lead to cystic bone formation and significant soft tissue swelling.

[0039] Alizarin Red S (ARS) characterization: The osteoblast mineralization nodule staining kit was used to detect the osteogenic induction activity of PEGS-OH / CPC / rhBMP-2. This experiment is similar to the ALP activity characterization, with only the operation on day 7 being different. The process before this is not repeated here. On day 7, cells were processed and characterized: First, the 24-well plate was washed 3 times with PBS solution (1 mL / well), and the BMSCs were fixed with the fixative solution in the kit (200 uL / well) at room temperature in the dark.S Incubate the cells for 20 minutes, then wash twice with PBS. Add an appropriate amount of Alizarin Red S staining solution and stain for 30 minutes at room temperature in the dark. Add an appropriate amount of PBS solution for qualitative imaging. For quantitative characterization, discard the PBS solution and add 500 μL of 10% cetylpyridinium chloride to each well. Incubate in the dark until the mineralized nodules dissolve. Transfer an equal amount of sample from each well to a 96-well plate and measure the OD at 562 nm. Figure 9 The quantitative characterization results of ARS are: Figure 11 This is the qualitative characterization result of ARS, and the conclusion is consistent with the ALP characterization conclusion.

[0040] The above description is merely a preferred embodiment of the present invention and does not limit the present invention in any way. Any person skilled in the art who, without departing from the scope of the present invention, makes any equivalent substitution, modification, or other changes to the technical solution and technical content disclosed in the present invention shall be deemed to be within the scope of the present invention and still fall within the scope of protection of the present invention.

Claims

1. A bone cement material, characterized in that: Bone morphogenetic protein-2 is doped into bone cement raw materials composed of polyester macromolecule PEGS-OH and calcium phosphate CPC, and the bone cement material is prepared under the action of a cross-linking agent.

2. The bone cement material with high osteogenic activity according to claim 1, characterized in that The mass ratio of the polyester polymer PEGS-OH, calcium phosphate CPC and bone morphogenetic protein-2 is 2 mg:8 mg:(1 ug-5 ug).

3. The bone cement material with high osteogenic activity according to claim 2, characterized in that The mass ratio of the polyester polymer PEGS-OH, calcium phosphate CPC and bone morphogenetic protein-2 is 2mg:8mg:5ug.

4. The method for preparing the bone cement material according to any one of claims 1 to 3, characterized in that: Polyester polymer PEGS-OH, calcium phosphate CPC and a cross-linking agent are mixed evenly, bone morphogenetic protein-2 is added, and the mixture is stirred to obtain bone cement slurry, i.e., bone cement material.

5. The method for preparing bone cement material according to claim 4, wherein: The cross-linking agent is first mixed evenly with bone morphogenetic protein-2.

6. The method for preparing bone cement material according to claim 4, characterized in that: The cross-linking agent is LDI cross-linking agent.

7. The use of the bone cement material according to any one of claims 1 to 3, comprising any one of the following: (i) preparing tissue defect filling materials; (ii) preparing materials to guide tissue regeneration; (iii) preparation of medical cosmetic materials; (iv) preparing 3D printing matrix materials; (v) Serves as a loading matrix or carrier for growth factors or drugs.

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