A bone cement material and method of preparation and use

Abstract

The application discloses a bone cement material, a preparation method and application thereof. The bone cement material is prepared from a bone cement raw material which is composed of polyethylene glycol (PEG) and calcium phosphate (CPC) and doped with bone morphogenetic protein-2 (BMP-2) under the action of a crosslinking agent. The bone cement material has the properties of slow and stable release, high osteogenic activity and the like under the condition of adding a small amount of BMP-2.

Description

Technical Field

[0001] This invention relates to bone cement materials, preparation methods, and uses, specifically to a bone cement material, preparation method, and uses. Background Technology

[0002] Bone defects caused by disease, accidental injury, and natural disasters are increasing daily, leading to a huge market demand for bone tissue repair materials. This also places increasingly higher demands on existing repair materials in terms of surgical procedures, mechanical properties, and bioactivity. Polymethyl methacrylate (PMMA) bone cement is the most widely used. However, PMMA bone cement's bioinertness, non-degradability, and the high temperatures (approximately 70°C) generated during the reaction process significantly affect its tissue repair effectiveness, limiting its use to a filler rather than guiding bone regeneration. Therefore, calcium phosphate (CPC) bone cement, which is more biocompatible, was developed, utilizing its immobilized delivery of bone morphogenetic protein-2 (rhBMP-2) to enhance osteogenic activity. However, this binding method is merely physical adsorption; a large burst release of BMP-2 occurs during the release process, resulting in excessively high protein concentrations in the early stages of bone repair and insufficient concentrations in later stages, ultimately leading to deformed bone spurs and incomplete repair.

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

[0004] Purpose of the invention: In order to solve the problem of poor osteogenic effect caused by high initial instantaneous concentration and uneven release of rhBMP-2 in the prior art, the purpose of this invention is to provide an injectable bone cement material with stable sustained release and high osteogenic activity by adding a very small amount of rhBMP-2, as well as its preparation method and uses.

[0005] Previous research by our team has shown that introducing polyethylene glycol-modified polysaccharide sebacate (PEGS) can effectively improve the brittleness and poor collapse resistance of CPC, but the influencing factors on osteogenic properties have not been thoroughly investigated. This invention focuses on the effect of bone morphogenetic protein-2 rhBMP-2 on the osteogenic properties of bone cement materials.

[0006] Technical solution: 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 obtained under the action of a crosslinking 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 μg-5 μg).

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

[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 salt CPC and crosslinking 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 bone morphogenetic protein-2.

[0011] Furthermore, the crosslinking agent is an LDI crosslinking agent.

[0012] A third aspect of the present invention provides the use of the bone cement material, including any of the following:

[0013] (i) Preparation of tissue defect filling materials;

[0014] (ii) Prepare materials to guide tissue regeneration;

[0015] (iii) Preparation of medical aesthetic materials;

[0016] (iv) Preparation of 3D printing matrix materials;

[0017] (v) As a loading matrix or carrier for growth factors or drugs.

[0018] The principle of this invention is as follows:

[0019] The sustained-release effect of organic compounds on BMP-2 ensures its long-term release while reducing the instantaneous concentration, preventing excessively high concentrations that could lead to side effects such as bone spurs, increased osteoclast formation, or suboptimal osteogenic outcomes. BMP-2 is inextricably linked to bone cement; it induces the differentiation of bone marrow mesenchymal stem cells into osteoblasts, significantly enhancing the osteogenic activity of PEGS / CPC injectable bone cement. However, osteoblasts alone are insufficient for bone regeneration; essential components like calcium and phosphorus are also necessary. Therefore, CPC in the bone cement provides a stable supply of calcium and phosphate salts, providing raw materials for bone regeneration. Furthermore, PEGS, composed of sebacic acid, glycerol, and polyethylene glycol, has been shown to participate in the tricarboxylic acid cycle, providing cells with adenosine triphosphate (ATP), thus providing energy and further promoting osteogenic processes. As a biodegradable and absorbable material, PEGS / CPC injectable bone cement cannot exist permanently in the body like PMMA. Therefore, enhancing its osteogenic activity to induce bone regeneration is of great importance.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] 1. A small amount of rhBMP-2 is sufficient to achieve good osteogenic activity. Adding a small amount of rhBMP-2, while maintaining the same appearance and fluidity, yields good sustained-release properties and osteogenic activity. Alizarin red mineralization deposition of rhBMP-2 loaded in bone cement (Figure C) and alkaline phosphatase staining (Figure D) experiments show that the osteogenic activity of the material gradually increases with increasing rhBMP-2 protein content. This is attributed to rhBMP-2, as a functional protein, promoting the differentiation of bone marrow mesenchymal stem cells into osteoblasts.

[0022] 2. Good sustained-release effect. Low 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 rhBMP-2 protein, thereby enhancing the immobilization rate of rhBMP-2 and achieving sustained release of rhBMP-2, which is beneficial to osteogenic formation. Especially in the early stage of release, the release amount is small, avoiding the unsatisfactory osteogenic effect caused by initial burst release.

[0023] 3. Reduced burst release. Due to the presence of PEGS, the bone cement of this invention forms a large number of hydrogen bonds with rhBMP-2, resulting in a sustained release. The release of rhBMP-2 within 2 weeks is only 50 ng, accounting for 5% of 1 μg, and the release curve then tends to flatten out. In contrast, the release process of pure CPC generally exhibits a large burst release, especially in the first day, with approximately 60% released within 2 weeks.

[0024] 4. The addition of different contents of rhBMP-2 did not have a significant effect on the cytotoxicity of the materials, and the cell compatibility of each group was higher than the 70% required by the national standard GB / T 16886.5.

[0025] 5. No change in appearance: The amount of rhBMP-2 added is very small compared to bone cement, so it does not significantly interfere with the various properties of bone cement. Attached Figure Description

[0026] Figure 1 The appearance morphology of PEGS / CPC bone cement with different contents of rhBMP-2 is shown in the figures. A: 400 mg bone cement with 1 μg rhBMP-2; B: 400 mg bone cement with 3 μg rhBMP-2; C: 400 mg bone cement with 5 μg rhBMP-2.

[0027] Figure 2 This is the result of a cytotoxicity test;

[0028] Figure 3To obtain the release curve of rhBMP-2 in bone cement over 2 weeks, 5ug of rhBMP-2 was added;

[0029] Figure 4 To obtain the release curve of rhBMP-2 in bone cement over 2 weeks, 3ug of rhBMP-2 was added;

[0030] Figure 5 To obtain the release curve of rhBMP-2 in bone cement over 2 weeks, 1 μg of rhBMP-2 was added;

[0031] Figure 6 To obtain the release curve of rhBMP-2 in bone cement over 2 weeks, 8 μg of rhBMP-2 was added;

[0032] Figure 7 To obtain the release curve of rhBMP-2 in bone cement over 2 weeks, 10 μg of rhBMP-2 was added.

[0033] Figure 8 The release curve of rhBMP-2 in bone cement over 2 weeks is shown. Plain CPC with 5 μg rhBMP-2 added (general control).

[0034] Figure 9 Quantitative analysis of alizarin red mineralization deposition of rhBMP-2 loaded in bone cement;

[0035] Figure 10 For quantitative analysis of alkaline phosphatase by staining;

[0036] Figure 11 Photograph of alizarin red mineralization sediments;

[0037] Figure 12 Photograph of alkaline phosphatase staining. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be further described below.

[0039] Example 1: Preparation of PEGS / CPC / rhBMP-2

[0040] A fixed mass ratio of polyester polymer PEGS-OH to calcium phosphate salt CPC of 2:8 was used for the bone cement, which falls between liquid and solid states and is a suitable injectable state. The following investigation explores the effects of adding different masses of rhBMP-2 on the osteogenic capacity and sustained-release effect of the bone cement.

[0041] Weigh out three portions each of 80mg PEGS-OH, 320mg CPC, and 26.4uL LDI, mix them thoroughly, 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 the bone cement still retains good fluidity after the addition of rhBMP-2. Figure 1 As can be seen, the bone cement has a viscous toothpaste-like consistency. Because the amount of BMP-2 added is extremely small, its amount has no significant impact on the state of the bone cement; its viscosity and flowability are good. However, as the amount increases, such as adding 8 μg of rhBMP-2, the flowability and viscosity deteriorate, rendering it non-injectable and unsuitable for use as a bone material.

[0042] Example 2: Characterization of the cytotoxicity of the bone cement of the present invention

[0043] To evaluate the cytocompatibility of bone cement, this study used mouse fibroblasts (L929) to culture in bone cement extract and investigated the cytotoxicity of the cells and extract after 24 hours of culture.

[0044] First, bone cement containing rhBMP-2 was prepared in a clean bench: 80 mg of PEGS-OH, 320 mg of CPC, and 1 μg, 2 μg, and 3 μg of rhBMP-2 lyophilized powder were weighed out and sterilized with UV light. The crosslinking agent LDI was sterilized by filtration. 26.4 μL of LDI was thoroughly mixed with rhBMP-2, and then mixed with PEGS-OH and CPC to form a bone cement slurry. The blank group did not contain rhBMP-2, resulting in four bone cements with different rhBMP-2 contents (B0, B1, B2, and B3). The mixture was allowed to fully solidify at 37°C for 24 hours. The extract was then prepared: 2 mL of DMEM culture medium was added to the bone cement at a ratio of 200 mg bone cement to 1 mL culture medium. Simultaneously, L929 cells were inoculated into 96-well plates (1×10⁻⁶ cells / well). 4 Six replicates were set up per well, and the samples were placed in a cell culture incubator with constant temperature and humidity of 95% air / 5% carbon dioxide. After 24 hours, the culture medium in the cells was discarded, and extraction buffer (100 μL / well) was added. The cells were then cultured for another 24 hours. The MTT assay was then performed on the samples according to the national standard GB / T 16886.5-2017 to detect the cytotoxicity of bone cement. Figure 2 The cytotoxicity results indicate that bone cement (group B0) itself has a certain degree of cytotoxicity, and the addition of rhBMP-2 (B1, B2, B3) has no significant effect on cytotoxicity, all of which meet the national standards.

[0045] Example 3 Characterization of the release kinetics of rhBMP-2 in the bone cement of the present invention

[0046] 1. Add 5ug rhBMP-2

[0047] The cumulative release curve of rhBMP-2 in bone cement was detected using an enzyme-linked immunosorbent assay (ELISA) kit. 80 mg PEGS-OH, 320 mg CPC, 26.4 μL LDI, and 5 μg rhBMP-2 lyophilized powder were weighed. First, rhBMP-2 was thoroughly mixed with LDI, then mixed with PEGS-OH and CPC, and stirred thoroughly to form a bone cement slurry.

[0048] The solution was filled into a 6 mm diameter, 6 mm deep polytetrafluoroethylene cylindrical negative mold. After complete curing at 37°C, the mold was removed. The bone cement was immersed in 2 mL of PBS solution. At pre-set time points, 2 mL of the sustained-release solution was collected, and then 2 mL of fresh PBS solution was added to continue the release. The sustained-release solutions collected at each time point were numbered and stored at -20°C. According to the ELISA kit instructions, the concentration of rhBMP-2 in the sustained-release solution was detected using the rhBMP-2 ELISA immunoassay kit, and the OD value at 450 nm was measured using a SPECTRAmax384 microplate reader. A curve equation was fitted based on the standard curve, and the release amount of rhBMP-2 at each time point was calculated.

[0049] Figure 3 The cumulative release curve of rhBMP-2 over 14 days shows that in the first 7.5 hours, rhBMP-2 has a small burst release, followed by a slowdown in the release rate. The overall release is relatively stable and can be sustained, which is beneficial for osteogenic growth and increased activity.

[0050] 2. Add 3ug rhBMP-2

[0051] The method is the same as 1, except that 3ug of rhBMP-2 lyophilized powder is added.

[0052] Figure 4 The cumulative release curve of rhBMP-2 over 14 days shows a rapid release in the first 11 hours, followed by a slower release rate, with a relatively stable overall release that is conducive to osteogenic growth and enhanced activity.

[0053] 3. Add 1ug rhBMP-2

[0054] The method is the same as 1, except that 1ug of rhBMP-2 lyophilized powder is added.

[0055] Figure 5 The cumulative release curve of rhBMP-2 over 14 days is shown. In the first 9 hours, rhBMP-2 is released rapidly, then the release rate slows down, and the overall release is relatively stable and continuous, which is beneficial to osteogenic and activity enhancement.

[0056] 4. Add 8ug rhBMP-2

[0057] The method is the same as 1, except that 8ug of rhBMP-2 lyophilized powder is added.

[0058] Figure 6 The cumulative release curve of rhBMP-2 over 14 days is shown. rhBMP-2 is released rapidly, reaching 2.5 μg within two weeks. Experiments indicate that the amount of rhBMP-2 added cannot be increased indefinitely. With increasing addition, the release amount and rate of rhBMP-2 increase, with a significant initial burst release, which is detrimental to osteogenic formation.

[0059] 5. Add 10ug rhBMP-2

[0060] The method is the same as 1, except that 10ug of rhBMP-2 lyophilized powder is added.

[0061] Figure 7 The cumulative release curve of rhBMP-2 over 14 days is shown. rhBMP-2 is released rapidly, reaching 3 μg within 2 weeks. Experiments indicate that the amount of rhBMP-2 added cannot be increased indefinitely. With increasing addition, the release amount and rate of rhBMP-2 increase, with a significant initial burst release, which is detrimental to osteogenic formation.

[0062] also,

[0063] Depend on Figure 3 , Figure 4 , Figure 5 It is known that the bone cement prepared by this invention has a relatively small release amount within 2 weeks. A small burst release occurred in the first day, followed by a more stable release. This reduced the concentration of BMP-2 protein in the early stage, ensured the stability of the concentration in the later stage, improved osteogenicity, and achieved a better repair effect.

[0064] Depend on Figure 6 , Figure 7 Comparative studies show that with increasing rhBMP-2 dosage, the initial burst release increases, followed by a faster release rate, reaching over 2.5 μg within two weeks. This results in uneven BMP-2 protein concentration release, affecting osteogenesis and repair, and causing ectopic osteogenesis and related soft tissue inflammation. Therefore, more rhBMP-2 is not necessarily better, and the dosage significantly impacts osteogenesis.

[0065] Depend on Figure 3 and Figure 8Comparative analysis shows that the release amount of bone cement prepared by this invention within 2 weeks is far less than that of ordinary pure CPC bone cement, and the initial burst release amount is significantly reduced compared to ordinary pure CPC bone cement. This indicates that the protein concentration of the osteogenic material prepared by this invention will not be too high in the early stage of bone repair, thus ensuring the concentration in the later stage, which is conducive to bone survival and repair.

[0066] Example 4 Characterization of the osteogenic induction activity of the bone cement of the present invention

[0067] ALP activity characterization: The osteogenic induction activity of PEGS-OH / CPC / rhBMP-2 was detected using an ALP kit. This study used rat bone marrow mesenchymal stem cells (BMSCs). S The ALP activity of samples was detected after 7 days of co-culturing with bone cement in 24-well plates. Six groups were set up, with three replicates per group. Each sample weighed 1-2 mg. The Blank group did not contain bone cement. Groups B0, B1, B2, B3, and B4 represented the addition of 0 μg, 1 μg, 2 μg, 3 μg, and 4 μg of rhBMP-2 to the bone cement, respectively. Samples were first subjected to UV irradiation before being co-cultured with BMSCs. S Co-culture was performed, and the cells were replaced with osteogenic induction medium prepared using the OriCell rat bone marrow mesenchymal stem cell osteogenic induction differentiation kit on days 1, 3, and 5, while being wrapped in aluminum foil to protect them from light. Qualitative characterization 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 min, then washed three times with PBS, and incubated with 200 μL / well of BCIP / NBT staining working solution for 30 min at room temperature in the dark before photography. Quantitative characterization of ALP was performed: 24-well plates were washed three times with PBS, lysed with 100 μL / well of NP-40 lysis buffer, and incubated at 37°C for 90 min. 96-well plates were prepared, and the samples, diluents, and chromogenic solutions were added. The plates were then incubated at 37°C in the dark for 30 min. Finally, 100 μL of reaction stop solution was added to each well, and the OD value at 405 nm was measured. Figure 10 The quantitative results for ALP show that as the rhBMP-2 content gradually increases, rhBMP-2 has a significant promoting effect on BMSCs. S It has osteogenic differentiation capacity, but its activity tends to plateau as the amount increases (e.g., above 5ug), and it can lead to cystic bone formation and significant soft tissue swelling. Therefore, the dosage of rhBMP-2 should not be increased arbitrarily. Figure 12 The results of ALP qualitative analysis showed 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 bone differentiation (B0 group). As the rhBMP-2 content increases, the bone-promoting ability of bone cement is greatly enhanced. However, as the amount increases (e.g., above 5ug), the activity tends to level off, and it will lead to cystic bone formation and significant soft tissue swelling.

[0068] Alizarin Red S (ARS) Characterization: The osteogenic induction activity of PEGS-OH / CPC / rhBMP-2 was detected using an osteoblast mineralization nodule staining kit. This experiment is similar to ALP activity characterization, with the only difference being the procedure on day 7. The procedures before this point will not be described in detail. On day 7, cells were treated and characterized: 24-well plates were washed three times with PBS solution (1 mL / well), and BMSCs were fixed with the fixative solution (200 μL / well) provided in the kit at room temperature in the dark. S Cells were stained for 20 min, then washed twice with PBS solution, and an appropriate amount of Alizarin Red S staining solution was added. Staining was performed at room temperature in the dark for 30 min. Afterwards, an appropriate amount of PBS solution was added for qualitative imaging. Quantitative characterization was then performed: the PBS solution was discarded, and 500 μL of 10% hexadecylpyridine chloride was added to each well. The cells were treated in the dark until the mineralized nodules dissolved. An equal volume of sample from each well was transferred to a 96-well plate, and the OD value at 562 nm was measured. Figure 9 The results are quantitative characterizations of ARS. Figure 11 The qualitative characterization results for ARS are consistent with the characterization results for ALP.

[0069] The above are merely preferred embodiments of the present invention and do not constitute any limitation on the present invention. Any equivalent substitutions or modifications made by those skilled in the art to the technical solutions and content disclosed in the present invention without departing from the scope of the present invention shall be deemed to have remained within the scope of protection of the present invention.

Claims

1. A bone cement material, characterized by, The bone cement material is prepared by mixing bone morphogenetic protein-2 with bone cement raw materials containing polyester macromolecule PEGS-OH and calcium phosphate salt CPC under the action of a crosslinking agent, wherein the mass ratio of polyester macromolecule PEGS-OH to calcium phosphate salt CPC is 80 mg:320 mg, and the addition amount of bone morphogenetic protein-2 is 1 μg to 5 μg based on the total mass of PEGS-OH and CPC.

2. The bone cement material according to claim 1, characterized in that, The addition amount of bone morphogenetic protein-2 is 5 μg.

3. A method for the production of a bone cement material according to claim 1 or 2, characterized in that The polyester macromolecule PEGS-OH, the calcium phosphate salt CPC and the crosslinking agent are mixed uniformly, and then the bone morphogenetic protein-2 is added and stirred to obtain the bone cement slurry.

4. The method of preparing a bone cement material according to claim 3, characterized in that, The crosslinking agent is first mixed uniformly with the bone morphogenetic protein-2.

5. The method of preparing a bone cement material according to claim 3, characterized in that, The crosslinking agent is an LDI crosslinking agent.

6. Use of the bone cement material according to claim 1 or 2, comprising any one of the following: (i) preparing a tissue defect filling material; (ii) preparing a guided tissue regeneration material; (iii) preparing a medical cosmetology material; (iv) preparing a 3D printing matrix material; (v) as a loading matrix or carrier material for preparing growth factors or drugs.

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