A biodegradable, injectable adhesive bone cement for osteoporosis, its preparation method and application

Abstract

The present invention provides a biodegradable, injectable adhesive bone cement for osteoporosis, its preparation method and application. The composition for preparing the injectable bone cement comprises a liquid phase component and a solid phase component, wherein the liquid phase component includes a polyester polymer; the solid phase component includes a phosphate or a phosphate and its derivatives. The injectable bone cement is formed by cross-linking reaction of a polyester polymer, a phosphate and a phosphate derivative, and can be injected, biodegradable, adhesive to bone tissue, capable of regulating the bone microenvironment and treating osteoporosis. The biosafe bone cement of the present invention can highly fit with complex tissue defect sites, has no obvious heat release phenomenon during the curing process, can achieve strong adhesion to bone tissue, and its composition and structure can promote bone tissue repair under osteoporosis pathological conditions and improve the quality of bone repair. It is a bone tissue repair material with clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the fields of materials science and medicine, and particularly relates to an injectable bone cement with self-crosslinking and hydration enhancement in vivo environment, and its preparation method and application. In particular, it is a filling material for repairing bone tissue defects in complex structures or osteoporotic pathological environments. Background Art

[0002] Bone loss, bone quality deterioration, and destruction of bone microstructure caused by osteoporosis are the main causes of osteoporotic bone defects. Minimally invasive treatment has become one of the main treatment methods for osteoporotic fractures due to its advantages such as convenient surgery, small trauma, and quick recovery. Among them, injectable materials such as polymethyl methacrylate (PMMA) bone cement and calcium phosphate cement (CPC) are widely used in clinical bone defect repair due to their good operability and sufficient mechanical properties. However, existing bone cements, as macroscopic materials, cannot penetrate to the cellular level and are difficult to regulate osteoporotic bone regeneration in situ and precisely, so they are only used as filling materials with low bioactivity, resulting in the need to improve the efficiency of bone defect repair. Therefore, in view of the microenvironment characteristics of osteoporotic bone defects, it is of great research significance to develop an injectable bone cement that can regulate the bone microenvironment in situ and precisely. In view of the tight network structure and mechanical strength of calcium phosphate inorganic substances and organic collagen in natural bone, it is of great significance to construct a biomimetic organic / inorganic composite injectable bone cement. Summary of the Invention

[0003] The purpose of the present invention is to provide a bone cement with biocompatibility, in vivo in-situ hydration and pore formation, high bioactivity, and can be formed by injection.

[0004] In the first aspect of the present invention, there is provided an injectable bone cement composition, which comprises:

[0005] (c1) a liquid phase component, which is a polyester polymer; and

[0006] (c2) a solid phase component, which is a phosphate or a combination of phosphate and phosphate derivatives.

[0007] In another preferred example, the injectable bone cement composition further comprises a crosslinking agent, and the crosslinking agent is an isocyanate crosslinking agent.

[0008] In another preferred example, the crosslinking agent is selected from the group consisting of L-lysine diisocyanate (LDI), hexamethylene diisocyanate, toluene diisocyanate, or a combination thereof.

[0009] In another preferred example, the mass ratio of the polyester polymer to the crosslinking agent is 1:0.25 - 2, preferably 1:0.25 - 0.5, more preferably 1:0.3 - 0.5, such as 1:0.47.

[0010] In another preferred example, the polyester polymer is polyhydroxy polyethylene glycolated polyglycerol sebacate PEGS-OH, and the amount of substance of the isocyanate groups in LDI is 0.5 - 2 times the amount of substance of the hydroxyl groups in PEGS-OH.

[0011] In another preferred example, the amount of substance of the isocyanate groups in LDI is 1 - 1.5 times the amount of substance of the hydroxyl groups in PEGS-OH, preferably 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4 times.

[0012] In another preferred example, the mass ratio of the liquid-phase component to the solid-phase component is 0.5:9.5 - 5:5.

[0013] In another preferred example, the mass ratio of the liquid-phase component to the solid-phase component is 1:9 - 9:1, preferably 2:8.

[0014] In another preferred example, the polyester polymer is selected from: polyglycerol sebacate, polyethylene glycolated polyglycerol sebacate, polyhydroxy polyethylene glycol, polyhydroxy polyethylene glycolated polyglycerol sebacate PEGS-OH,

[0015] Among them, the structural unit of polyhydroxy polyethylene glycolated polyglycerol sebacate PEGS-OH is shown as formula Ia or formula Ib:

[0016]

[0017] Among them, in formula Ia, m1 is an integer from 1 to 20, and n1 is an integer from 5 to 60;

[0018] In formula Ib, m2 is an integer from 1 to 20, and n2 is an integer from 5 to 60.

[0019] In another preferred example, in formula Ia, m1 is an integer from 2 to 18, an integer from 5 to 15, an integer from 2 to 8, an integer from 8 to 10. In another preferred example, n1 is an integer from 10 to 50, an integer from 20 to 40, an integer from 30 to 50, an integer from 8 to 35.

[0020] In another preferred example, in formula Ib, m2 is an integer from 2 to 18, an integer from 5 to 15, an integer from 2 to 8, an integer from 8 to 10. In another preferred example, n2 is an integer from 10 to 50, an integer from 20 to 40, an integer from 30 to 50, an integer from 8 to 35.

[0021] In another preferred example, the number-average molecular weight of the polyester polymer is 3.0 - 100.0 kDa, preferably 6.0 - 50.0 kDa, more preferably 7.0 - 40.0 kDa.

[0022] In another preferred example, the dispersity coefficient of the polyester polymer is 1.1 - 2.0, preferably 1.2 - 1.7;

[0023] In another preferred example, in one structural unit, the polyester polymer contains on average at least 2 -OH groups.

[0024] In another preferred example, the phosphate is selected from: tetracalcium phosphate, calcium dihydrogen phosphate, tricalcium phosphate, or a mixture of two or more thereof.

[0025] In another preferred example, the phosphate derivative is selected from: sodium etidronate, sodium clodronate, sodium pamidronate, sodium tiludronate, sodium alendronate, sodium neridronate, sodium olpadronate, sodium risedronate, sodium ibandronate, zoledronic acid, or a mixture of two or more thereof.

[0026] In another preferred example, the phosphate derivative is sodium alendronate.

[0027] In another preferred example, the mass fraction of the phosphate derivative is 0.001% - 10%, preferably 0.005% - 5%, more preferably 0.01% - 5%.

[0028] In another preferred example, the polyester polymer is prepared by the following method:

[0029] (S1) Provide sebacic acid, polyethylene glycol diglycidyl ether, and a catalyst;

[0030] (S2) In a first solution, mix sebacic acid, polyethylene glycol diglycidyl ether, and the catalyst, and react to obtain the polyester polymer.

[0031] In another preferred example, the method is carried out under an atmosphere of high-purity nitrogen or high-purity argon.

[0032] In another preferred example, the molecular weight of the polyethylene glycol diglycidyl ether is 500 Da.

[0033] In another preferred example, the molar ratio of sebacic acid to polyethylene glycol diglycidyl ether is 5:1 - 1:5.

[0034] In another preferred example, the catalyst is selected from bis(tetrabutylammonium hydroxide) sebacate.

[0035] In another preferred example, the first solution includes anhydrous N,N-dimethylformamide.

[0036] In another preferred example, the reaction of the method is carried out at 90 - 110 °C, preferably 95 - 105 °C, more preferably 92 - 103 °C, for example 100 °C.

[0037] In another preferred example, the reaction time is 65 - 80 h, preferably 68 - 78 h, more preferably 70 - 74 h, for example 72 h.

[0038] In another preferred example, the method further comprises a post-treatment step. Preferably, the post-treatment includes sedimentation, drying, and dialysis purification.

[0039] In another preferred example, the sedimentation is carried out in ether.

[0040] In another preferred example, the drying is carried out by vacuum drying at room temperature for 5 - 20 h.

[0041] In another preferred example, the dialysis purification is carried out using a dialysis bag with a molecular weight cut-off of 3500 Da.

[0042] The present invention realizes high-strength mechanical support at the bone defect site through an organic / inorganic interpenetrating network system, and based on the chemical / physical dual bonding effect, realizes the strong bonding between the material and the bone. Finally, the bone cement precisely regulates the microenvironment for the regeneration of osteoporotic pathological bone. The present invention designs an organic / inorganic composite injectable bone cement with in-situ regulation of the bone microenvironment and bone bonding effect through a biofabrication strategy, meeting the requirements for the repair of osteoporotic bone defects and having great clinical significance.

[0043] In the second aspect of the present invention, there is provided an injectable bone cement, which is obtained by mixing the liquid-phase component and the solid-phase component of the injectable bone cement composition described in the first aspect and an optional cross-linking agent.

[0044] In another preferred example, the liquid-phase component and the solid-phase component are thoroughly and uniformly mixed to obtain a white dough-like slurry. The cross-linking agent is uniformly mixed with the slurry to obtain the injectable bone cement.

[0045] In another preferred example, the injectable bone cement is filled into a syringe and injected into the relevant site, thereby solidifying to form the bone cement described in the fourth aspect of the present invention.

[0046] In another preferred example, the storage modulus of the injectable bone cement during injection is 1×10 3 -4×10 4 Pa, preferably 10 4 Pa.

[0047] In another preferred example, the loss modulus of the injectable bone cement during injection is 2×10 3 -4×10 5Pa, preferably 10 4 Pa.

[0048] In another preferred example, the storage modulus is not higher than the loss modulus, meeting the injectability requirements.

[0049] In another preferred example, after the liquid-phase component and the solid-phase component are mixed, they are mixed with a cross-linking agent at 25°C - 40°C, more preferably at 35°C - 37°C, and optimally at 37°C.

[0050] In another preferred example, the liquid-phase component, the solid-phase component, and the cross-linking agent are as described above.

[0051] In the third aspect of the present invention, there is provided a method for preparing an injectable bone cement as described in the second aspect of the present invention, comprising the following steps:

[0052] The liquid-phase component, the solid-phase component, and optionally the cross-linking agent of the injectable bone cement composition described in the first aspect are mixed to obtain the injectable bone cement.

[0053] In another preferred example, after the liquid-phase component and the solid-phase component are mixed, they are mixed with a cross-linking agent at 25°C - 40°C, more preferably at 35°C - 37°C; optimally at 37°C.

[0054] The liquid-phase component, the solid-phase component, and the cross-linking agent are as described above.

[0055] In the fourth aspect of the present invention, there is provided a bone cement, which is obtained by curing the injectable bone cement described in the second aspect of the present invention.

[0056] In another preferred example, the curing time of the bone cement is 15 - 100 min, preferably 15 - 40 min, more preferably 12 - 20 min, such as 13 min, 17 min.

[0057] In another preferred example, the bone cement is cured at 25 - 40°C, preferably at 30 - 40°C, such as 37°C.

[0058] In another preferred example, the bone cement is obtained by injecting the injectable bone cement filled in a syringe into a relevant site and curing it in vivo. In another preferred example, the lap shear strength of the bone cement to bone tissue is 150 - 280 kPa.

[0059] In another preferred example, the bone cement has an inhibitory effect on osteoclasts.

[0060] In another preferred example, the bone cement has in vitro osteogenic properties.

[0061] In another preferred example, the bone cement has excellent biocompatibility.

[0062] In another preferred example, the bone cement has one or more of the following properties:

[0063] (p1) Increase the ratio of new bone volume;

[0064] (p2) Reduce the trabecular bone separation;

[0065] (p3) Cure rapidly and without heat generation during the curing process;

[0066] (p4) Have high strength and elasticity.

[0067] In a fifth aspect of the present invention, there is provided a kit for preparing the injectable bone cement described in the second aspect, the kit comprising:

[0068] (a) A first container and a liquid phase component located within the container;

[0069] (b) A second container and a solid phase component located within the container;

[0070] (c) A third container and a crosslinking agent located within the container;

[0071] In the first container, the liquid phase component is present in the form of a viscous liquid;

[0072] In the second container, the solid phase component is present in the form of a powder;

[0073] In the third container, the crosslinking agent is present in the form of a liquid,

[0074] wherein the liquid phase component, the solid phase component, and the crosslinking agent are as described above.

[0075] In a sixth aspect of the present invention, there is provided the use of the injectable bone cement composition described in the first method of the present invention, the injectable bone cement described in the second aspect, or the kit described in the fifth aspect of the present invention, for any one or two or more of the following applications:

[0076] (i) Preparing a tissue defect filling material;

[0077] (ii) Preparing a material for guided tissue regeneration;

[0078] (iii) Preparing a medical aesthetic material;

[0079] (iv) A 3D printing matrix material;

[0080] (v) A loading matrix or carrier for growth factors or drugs.

[0081] In another preferred example, for preparing a material for treating spinal fusion fractures.

[0082] In another preferred example, it is used to prepare materials for treating osteoporotic bone defects.

[0083] In the seventh aspect of the present invention, a method for filling tissue defects and / or guiding tissue regeneration is provided. The method includes applying the injectable bone cement described in the second aspect of the present invention to the site to be treated.

[0084] In the eighth aspect of the present invention, a method for loading growth factors and / or drugs and / or cells is provided. The method includes mixing the injectable bone cement described in the second aspect of the present invention with the growth factors and / or drugs and / or cells to be loaded.

[0085] In the ninth aspect of the present invention, a method for 3D printing a composite scaffold is provided. The method includes using the injectable bone cement described in the second aspect of the present invention as a 3D printing matrix material.

[0086] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Description of the Drawings

[0087] Figure 1 Shows the synthesis route of the polyhydroxy PEGS polymer in the present invention (A); the proton nuclear magnetic resonance spectrum of the polyhydroxy PEGS polymer (B).

[0088] Figure 2 Shows the Fourier transform infrared spectrum of the injectable composite bone cement in the present invention (A); the injectability of the main formula injectable bone cement (B); the rheological curve of the injectable bone cement at 37°C (C).

[0089] Figure 3 Shows the anti-fatigue performance of the injectable bone cement in the present invention (A); the compressive modulus of the injectable bone cement (B); the adhesion curve of the injectable bone cement (C); the lap shear strength of the injectable bone cement (D); the scanning electron microscope image of the injectable composite bone cement (E).

[0090] Figure 4 Shows the cytotoxicity of the injectable bone cement in the present invention (A); the in vitro performance of inhibiting osteoclasts of the injectable bone cement (B); the performance of promoting osteogenic differentiation of mesenchymal stem cells of each formula injectable bone cement (C).

[0091] Figure 5Shows the CT reconstruction images (A) after bone tissue repair with the optimal formulation injectable bone cement of the present invention compared with other materials; the new bone volume ratio (B) after bone tissue repair with the optimal formulation injectable bone cement compared with other materials; the trabecular separation (C) after bone tissue repair with the optimal formulation injectable bone cement compared with other materials. Detailed implementation mode

[0092] Through extensive and in - depth research, the inventor of the present invention unexpectedly discovered for the first time an injectable bone cement with excellent mechanical properties, adhesion properties, rapid in - vivo curing, and the ability to regulate the osteoporotic bone microenvironment.

[0093] Specifically, the bone cement introduces biodegradable polyhydroxy - polyethylene glycolated polyglycerol sebacate (PEGS - OH) as the organic phase of the bone cement, and phosphates and their derivatives as the inorganic phase, constructing an organic / inorganic composite injectable bone cement system based on the cross - linking of isocyanate with hydroxyl and amino groups. The bone cement has excellent injection performance, can rapidly cure and form in the in - vivo environment, and the cross - linking process is mild, without obvious heat release, and has no obvious toxicity to the host organism, and can be used for the filling of osteoporotic bone defects and the guided regeneration of bone tissue.

[0094] The bone cement of the present invention not only has excellent mechanical properties, bonding properties, and biological activity, as well as good cell compatibility and bone - guiding properties, but also has ideal tissue repair effects and clinical applicability, making the implantation of this material without open surgery, facilitating the operation of doctors while reducing the pain of patients, and providing new alternative materials and design ideas for minimally invasive treatment of tissue regeneration technology. The present invention is completed on this basis.

[0095] Terms

[0096] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0097] As used herein, when referring to a specifically recited numerical value, the term "about" means that the value can vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0098] As used herein, the term "comprising" or "including" can be open - ended, semi - closed, and closed. In other words, the term also includes "consisting essentially of...", or "consisting of...".

[0099] As used herein, the terms "PEG" and "polyethylene glycol" are used interchangeably.

[0100] As used herein, the terms "PEGS", "PEGylated polyglycerol sebacate", "polyethylene glycolylated polyglycerol sebacate", and "PEGS polymer" are used interchangeably.

[0101] As used herein, the terms "CPC" and "phosphate and its derivatives" are used interchangeably.

[0102] As used herein, the terms "LDI" and "L-lysine diisocyanate" are used interchangeably.

[0103] As used herein, the terms "PEGS-OH" and "polyhydroxy PEGS" are used interchangeably.

[0104] As used herein, the terms "ALN" and "sodium alendronate" are used interchangeably.

[0105] As used herein, in the terms "PCA1", "PCA2", and "PCA3", P represents PEGS, C represents CPC, A represents phosphate derivatives, and 1, 2, and 3 refer to the percentage of the mass of the phosphate derivative in the total mass of the bone cement.

[0106] Polyhydroxy polyethylene glycolylated polyglycerol sebacate (PEGS-OH)

[0107] In the present invention, the polyhydroxy polyethylene glycolylated polyglycerol sebacate (PEGS-OH) has the structure shown in Formula Ia or Formula Ib:

[0108]

[0109] In Formula Ia, m1 is an integer from 1 to 20, and n1 is an integer from 5 to 60;

[0110] In Formula Ib, m2 is an integer from 1 to 20, and n2 is an integer from 5 to 60.

[0111] Specifically, the preparation method of the polyhydroxy polyethylene glycolylated polyglycerol sebacate includes the following steps:

[0112] (a1) Under the protection of an argon or nitrogen atmosphere, a diacid and a diglycidyl ether with a molar ratio of 1:1 are dissolved in a solvent (wherein the diacid can be sebacic acid, carboxylated PEG with a molecular weight of 250 Da, or carboxylated PEG with a molecular weight of 600 Da; the diglycidyl ether can be sebacic acid diglycidyl ether or polyethylene glycol diglycidyl ether with a molecular weight of 500 Da), and a bis-tetra-n-butylammonium hydroxide salt catalyst with a molar ratio of 0.6 mol% is added (wherein the carboxylic acid part of the ammonium salt can be sebacic acid, carboxylated PEG with a molecular weight of 250 Da, or carboxylated PEG with a molecular weight of 600 Da), and the reaction is carried out at 90 - 120 °C (more preferably 100 °C) for 1 - 3 days;

[0113] (b1) After the product of step (a1) is precipitated in diethyl ether and then vacuum dried at room temperature for 6 - 12 hours (more preferably 10 - 12 hours), polyhydroxy poly(ethylene glycol) - grafted polyglycerol sebacate polymer PEGS - OH is obtained.

[0114] In another preferred embodiment, the preparation method further comprises the step;

[0115] (c1) Purify the PEGS - OH obtained in step (b1) by dialysis to obtain purified PEGS - OH.

[0116] The bone cement of the present invention

[0117] Bone cements are a class of biomedical materials that can play a supporting role at the site of tissue defects. The present invention provides a bone cement with biocompatibility, in - vivo hydration and pore - formation, and can be formed by injection.

[0118] As used herein, the terms "injectable bone cement" and "the bone cement of the present invention" can be used interchangeably.

[0119] The bone cement of the present invention is formed by mixing and reacting a liquid phase component, a solid phase component and an optional cross - linker; the liquid phase component is a polyester polymer; the solid phase component is a phosphate or a combination of a phosphate and its derivatives; the cross - linker is an isocyanate - type cross - linker.

[0120] In another preferred embodiment, the liquid phase component includes polyhydroxy poly(ethylene glycol) - grafted polyglycerol sebacate;

[0121] The solid phase component includes tetracalcium phosphate, anhydrous calcium phosphate and alendronate sodium;

[0122] The cross - linker includes L - lysine diisocyanate.

[0123] In a preferred embodiment, polyhydroxy poly(ethylene glycol) - grafted polyglycerol sebacate (PEGS - OH) is used as the organic phase, phosphate (CPC) and its derivative alendronate sodium (ALN) are used as the inorganic phase, and they are cross - linked and cured by L - lysine diisocyanate (LDI) and injected into the relevant site by syringe injection to obtain a bone cement material with certain mechanical properties.

[0124] The mass ratio of the liquid phase component to the solid phase component is 1 - 3:9 - 7, preferably 2:8.

[0125] In another preferred embodiment, the phosphate and its derivatives and the PEGS - OH are combined by physical mixing.

[0126] The PEGS - OH of the present invention and the cross - linker are combined by an addition reaction between the hydroxyl group of PEGS - OH and the isocyanate group of LDI.

[0127] The mechanical properties and rheological properties of the bone cement according to the present invention can be adjusted and optimized by the mass ratio of polyhydroxy polyethylene glycolated polyglycerol sebacate to phosphates and their derivatives.

[0128] In another preferred embodiment, the mass ratio of the polyester polymer to the crosslinking agent can be used to adjust the modulus, rheological properties and pore content of the bone cement.

[0129] In another preferred embodiment, the mass ratio of the polyester polymer to phosphates and their derivatives can be used to adjust the rheological properties, setting time, cytotoxicity and osteogenic properties of the bone cement.

[0130] The bone cement has one or more of the following characteristics:

[0131] (1) The compressive modulus is between 5 and 50 MPa, preferably between 10 and 20 MPa;

[0132] (2) At 37 °C, the setting time is between 10 and 40 min, preferably between 15 and 20 min;

[0133] (3) At 25 °C, the setting time is greater than 25 min.

[0134] The cytotoxicity of the injectable bone cement is 90% of that of the blank control group without adding any materials.

[0135] The injectable bone cement does not break or crumble under a pressure of 5000 N.

[0136] The surface of the injectable bone cement has a porous structure with different sizes.

[0137] The injectable bone cement does not disperse under the impact of water flow at a normal flow rate of 5 - 8 L / min.

[0138] There is no obvious exothermic phenomenon during the curing process of the injectable bone cement.

[0139] The injectable bone cement has strong bonding properties to bone tissue, and the lap shear strength is between 150 - 280 kPa.

[0140] The injectable bone cement has an obvious killing effect on osteoclasts derived from bone marrow macrophages.

[0141] The injectable bone cement has an obvious osteogenic differentiation effect on bone marrow mesenchymal stem cells.

[0142] Preparation method of the bone cement of the present invention

[0143] The present invention provides a preparation method of a PEGS-OH, CPC, ALN composite bone cement, comprising the following steps:

[0144] (i) Provide polyhydroxy polyethylene glycolated polyglycerol sebacate (PEGS-OH), calcium phosphate salt (CPC), phosphate derivative (ALN), and L-lysine diisocyanate (LDI).

[0145] (ii) After mixing PEGS-OH and CPC, add LDI and ALN to obtain the bone cement slurry.

[0146] Transfer the above bone cement slurry into a syringe barrel, and it can be injected into the defect site to form bone cement by in vivo curing.

[0147] In another preferred example, in the step (i), the molecular weight of PEGS-OH (polymer) is 12000 Da, the molecular weight of L-lysine diisocyanate (LDI) is 226.23 g / mol, and the molecular weight of alendronate sodium (ALN) is 272.09 g / mol.

[0148] In the step (ii), the mass ratio of CPC to ALN is 20 - 80:1 - 4.

[0149] In another preferred example, in the step (ii), the mass ratio of the liquid phase to the solid phase feed is 1 - 3:7 - 9.

[0150] In another preferred example, in the step (ii), the amount of substance of the isocyanate group in LDI is 1.5 - 1.0 times the amount of substance of the hydroxyl group in PEGS-OH.

[0151] In another preferred example, in the step (ii), the reaction temperature is 37 °C and the reaction duration is 15 - 30 min.

[0152] The curing time of the injectable bone cement shortens with the increase of the crosslinking agent content, and the curing time can be adjusted between 15 - 40 min. The compressive modulus of the bone cement increases with the increase of the CPC and ALN contents, and the initial compressive modulus can be adjusted between 5 - 18 MPa.

[0153] The main advantages of the present invention include:

[0154] 1) The bone cement described in the present invention has excellent injectability and mechanical strength;

[0155] 2) The mixture of the bone cement described in the present invention can be in-situ cured by injection to the desired site, and its curing time can be adjusted between 15 - 40 min;

[0156] 3) The rheological properties, injection properties, mechanical properties, and cell behavior of the bone cement described in the present invention can be regulated and optimized by changing the component contents.

[0157] 4) The bone cement of the present invention can freely fill the defect site and can fit well with the height of the defect;

[0158] 5) The bone cement of the present invention has biodegradability;

[0159] 6) The bone cement of the present invention has good bone adhesion performance and can better improve the adhesion effect between the bone cement and osteoporotic bone tissue;

[0160] 7) The cross-linking process of the bone cement of the present invention is mild and there is no obvious heat release;

[0161] 8) Inorganic CPC in the bone cement of the present invention can guide bone tissue regeneration and regulate the pathological microenvironment of osteoporosis;

[0162] 9) Inorganic ALN in the bone cement of the present invention can repair osteoporotic bone tissue and promote bone tissue regeneration;

[0163] 10) The minimally invasive property of the bone cement of the present invention makes it have considerable prospects in clinical applications;

[0164] 11) PEGS used in the bone cement of the present invention is a highly biocompatible polymer, and PGS derived from PEGS has been approved by the FDA for clinical use and has no potential biological toxicity.

[0165] The following further elaborates the present invention in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0166] Example 1. Synthesis of Polyhydroxy PEGS

[0167] (a) Weigh 1.95 g of sebacic acid (0.01 mol) and 20 g (0.07 mol) of tetrabutylammonium hydroxide (25 wt% aqueous solution) and add them to 100 mL of 95% ethanol. Stir and react at 55 °C for 30 min, then rotary evaporate to remove ethanol at 40 °C, and finally lyophilize to obtain the product tetrabutylammonium sebacate (TBAS);

[0168] (b) Under the protection of a high-purity nitrogen or high-purity argon atmosphere, weigh 20.23 g of sebacic acid (0.1 mol), 50.00 g of polyethylene glycol diglycidyl ether (0.1 mol) and 0.41 g of the product TBAS from step (a). Blend and dissolve the above raw materials in 180 mL of anhydrous N,N-dimethylformamide and stir and react at 100 °C for 72 h;

[0169] (c) The product obtained in step (b) was precipitated in diethyl ether and then dried in vacuo at room temperature for 12 hours;

[0170] (d) The product of step (c) was purified by dialysis using a dialysis bag with a molecular weight cut-off of 3500 Da to obtain purified linear polyhydroxy PEGs.

[0171] The schematic diagram of the synthesis reaction is as shown in Figure 1 A shown in

[0172] The 1H NMR spectrum of the obtained polyhydroxy PEGs polymer is as shown in Figure 1 B shown in

[0173] Example 2. Characterization of PEGS-OH / CPC and ALN Composite Bone Cement

[0174] Explore the composite situation of polyester polymer PEGS-OH / CPC and ALN at different ratios.

[0175] Prepare PEGS-OH by the method of Example 1. Weigh 200 mg of PEGS-OH and 800 mg of CPC, mix them evenly, then add 70.56 mg, 78.40 mg, 86.24 mg, 94.08 mg of cross-linking agent LDI to the above slurry, and at the same time add 0 mg, 10 mg, 20 mg, 30 mg of ALN respectively and mix evenly to form bone cement slurries PC, PCA1, PCA2, PCA3.

[0176] The above slurries were tested by Fourier transform infrared spectroscopy using the potassium bromide tablet method. As can be seen from Figure 2 A in

[0177] As shown in Figure 2 B, the bone cement slurries of the main formulations PCA1, PCA2, PCA3 all have good injectability.

[0178] The rheological properties of the above were tested by a rotational rheometer to study their changes with frequency. The use process of the rotational rheometer includes the following steps:

[0179] (a1) Turn on the rotational rheometer (Thermo Hakke, USA), set the temperature to (37 °C) and control the temperature, set the frequency at 10 Hz, and set the strain at 1.0%;

[0180] (b1) Prepare bone cement slurries (PC, PCA1, PCA2, PCA3) with different composite ratios according to the above method;

[0181] (c1) filling the bone cement slurry in (b1) into the syringe barrel for standby use;

[0182] (d1) Use the syringe in (c1) to quickly inject into the 20 mm diameter steel parallel plate and start the test immediately;

[0183] (e1) Record the changes of storage modulus (G') and loss modulus (G") over time.

[0184] from Figure 2 As can be seen in C, the loss modulus is greater than the storage modulus, indicating fluidity, which means that the bone cement slurry tends to be in a liquid state and is a suitable injectable state.

[0185] The results showed that compared with the PC group, the bone cements in the three groups of PCA1, PCA2 and PCA3 had more suitable clinical window periods and appropriate injectability.

[0186] Example 3. Mechanical Properties and Bone Adhesion Properties of the Bone Cement of the Invention

[0187] Weigh 200 mg of PEGS-OH and 800 mg of CPC, mix them evenly, then add 70.56 mg, 78.40 mg, 86.24 mg, and 94.08 mg of cross-linking agent LDI to the above slurry, and add 0 mg, 10 mg, 20 mg, and 30 mg of ALN, respectively, recorded as PC, PCA1, PCA2, and PCA3. After sufficient stirring, fill them into a polytetrafluoroethylene cylindrical mold with a diameter of 10 mm and a depth of 6 mm. The fatigue resistance of bone cement was investigated by cyclic compression experiments. Program control, set the compression rate to 10 mm / min. After compression to a stress of 12 MPa, release the stress to 0 MPa at a rate of 10 mm / min, which is one cycle. Cycle compression 10 times, record the stress-strain curve, Figure 3 Figure A shows that the bone cement of the present invention has excellent elasticity because the PEGS-OH component is an elastic polymer. After 10 cycles of compression, the bone cement has good recovery performance, which is not possessed by rigid materials. In addition, when in use, bone cement is mainly subjected to longitudinal pressure, so the compression test is used to examine the compressive properties of bone cement. A universal testing machine was used to study the compressive properties of bone cement. The cylindrical bone cement sample was placed flat in the center of the compression platform, and parameters such as the sample diameter and the original gauge length were input. A 5kN sensor was selected, and five parallel samples were set for each group of samples to be tested. During the compression test, the compression rate was 10mm / min until the force reached 5000N, and the stress-strain curve was recorded. The results are as follows. Figure 3 As shown in Figure B, with the increase of ALN and LDI content, the mechanical properties of bone cement increased, and the PCA1, PCA2, and PCA3 groups were significantly better than the PC group. This was due to the improved mechanical properties of the formed interpenetrating network.

[0188] The lap shear test was used to check the bone adhesion performance of bone cement. Fresh pig bone blocks were purchased and the surface was polished with sandpaper. Four samples of PC, PCA1, PCA2, and PCA3 were prepared according to the above method. They were fully stirred and bone cement was injected between the bone pieces to achieve overlapping bonding. They were cured at an ambient temperature of 37°C and then tensile tested using an electronic mechanical testing machine equipped with a 5kN force sensor to record the force-displacement curve. In addition, the lap shear strength was calculated using the following formula: lap shear strength = maximum force during shearing process / (width of adhesion area × length of adhesion area). Figure 3 As shown in C and D, with the increase of ALN and LDI content, the adhesion ability and lap shear strength of bone cement continued to increase. The PCA1, PCA2, and PCA3 groups were significantly better than the PC group. The shear process failure force of the PCA3 group could reach 120N, and the interface shear strength could reach 270kPa, which could achieve effective adhesion to bone tissue.

[0189] After the above screening, PCA3 was basically determined to be the best formula for mechanical properties in each group. The microscopic morphology of the best formula bone cement is as follows Figure 3 As shown in E, a porous structure that facilitates tissue ingrowth appeared inside the bone cement. Compared with the PC group, the PCA3 group had larger pores and better connectivity. This porous structure has a positive effect on cell adhesion and tissue ingrowth during tissue regeneration.

[0190] Example 4. Osteoclast Inhibitory and Osteogenic Properties of the Bone Cement of the Invention in Vitro

[0191] In order to evaluate the cell compatibility of bone cement, this study used mouse fibroblasts (L929) and bone cement co-culture to investigate cytotoxicity according to the national standard "GB / T 16886.5-2017". Weigh 200 mg of PEGS-OH and 800 mg of CPC, mix them evenly, add 70.56 mg, 78.40 mg, 86.24 mg, and 94.08 mg of crosslinker LDI LDI to the above slurry, and add 0 mg, 10 mg, 20 mg, and 30 mg of ALN, respectively, recorded as PC, PCA1, PCA2, and PCA3. After sufficient stirring, fill it into a polytetrafluoroethylene cylindrical mold with a diameter of 10 mm and a depth of 6 mm, and select and add commercial PMMA bone cement (Spineplex, ) was used as the PMMA control group, and no material was added as the blank control group. According to the national standard GB / T 16886.5-2017 testing requirements, an appropriate amount of L929 cells were inoculated on each group of samples and co-cultured with the materials, and then the MTT experiment was performed to detect the cytotoxicity of bone cement. Figure 4The cytotoxicity results of A indicate that while the cytotoxicity of the bone cements in the PC, PCA1, PCA2, and PCA3 groups meets the national standards, it is significantly lower than that of the commercial PMMA bone cement.

[0192] The anti-resorptive properties of the optimal formulation bone cement were evaluated by tartrate-resistant acid phosphatase (TRAP) staining and F-actin ring immunofluorescence staining. The groups were set up according to the above method, and the samples were prepared. Bone marrow macrophages (BMMs) were seeded at a density of 1×10 5 / well, with 5 parallel samples in each group. The pre-prepared α-MEM medium (containing 100 ng / mL of RANKL and 60 ng / mL of M-CSF) was added and co-cultured with the cells. After 7 days of culture, the medium was aspirated, and 2.5% glutaraldehyde solution was added to fix the cells in preparation for TRAP staining. For TRAP staining, after washing 3 times with PBS, 200 μL of TRAP staining solution was added to each well in the dark environment, and incubated at 37 °C in the dark for 30 min. After completion, the TRAP staining solution was aspirated, and washed 3 times with PBS. An inverted microscope was used to observe the cells, and the cells containing more than 3 nuclei were osteoclasts, and the photos were taken. The results are as Figure 4 shown in Figure B. Compared with other groups, the number of osteoclasts in the PCA3 group was significantly reduced, indicating that the anti-resorptive property of the PCA3 bone cement is the most superior compared with other groups.

[0193] The osteogenic properties of the bone cement were evaluated by alkaline phosphatase (ALP) activity and mineralization. The groups were set up according to the above method, and the samples were prepared. Bone marrow mesenchymal stem cells (BMSCs) were seeded at a density of 2×10 4 / well, with 5 parallel samples in each group, and cultured in a commercial osteogenic induction medium (RAXMX-90021, ) (α-MEM containing 2% FBS, 0.05 mM L-ascorbic acid, 100 mM β-glycerophosphate, and 100 mM dexamethasone). After 14 days of culture, the medium was removed, washed 2 times with PBS, and the cells were stained with a BCIP / NBT alkaline phosphatase color development kit and photographed. The results are as Figure 4 shown in Figure C. Each group had good in vitro osteogenic properties.

[0194] Example 5. Characterization of the Tissue Repair Effect of the Bone Cement of the Invention

[0195] To evaluate the in vivo tissue repair effect of the bone cement, a rat femoral defect repair model was selected in this study to compare the bone regeneration promoting abilities of the commercial PMMA bone cement and the bone cement of the present invention.

[0196] Female SD rats were raised to 6 months of age and underwent bilateral ovariectomy (OVX) surgery. According to the requirements of surgical operations, all surgical instruments were sterilized in advance with high temperature and high pressure, and the operation was performed under sterile conditions. The rats were anesthetized by intraperitoneal injection of 1wt% sodium pentobarbital at a dose of 40mg / kg rat body weight, fixed in the lateral position, shaved on the back and both sides of the abdomen and disinfected with iodine, and the skin and muscle layer were cut along the lateral abdomen to expose the adipose tissue. Use tweezers to push aside the fat mass to find the pink cauliflower-like ovarian tissue, use silk thread to ligate and remove the ovary between the fallopian tube and the ovary, and the contralateral ovary is removed in the same way. 12 weeks after OVX surgery, the rats gradually lost bone mass and developed into an osteoporosis model.

[0197] Osteoporotic SD rats were selected and anesthetized. The distal end of the femur was depilated and the skin was cut to expose the middle part of the femur. A circular trephine with a diameter of 3 mm was used to make a defect on the side of the femur for standby use. 200 mg of PEGS-OH and 800 mg of CPC were weighed and mixed evenly. 70.56 mg and 78.40 mg of cross-linking agent LDI were added to the above slurry, and 0 mg and 30 mg of ALN were added at the same time, respectively, recorded as PC and PCA3 groups. After sufficient stirring, they were injected into the above femoral defect and recorded as the experimental group. In addition, a commercially available PMMA product was selected to be implanted in the above femoral defect as a PMMA control group, and a blank control group was used without filling any material, and then the skin was sutured.

[0198] After 6 weeks, the rats were killed by overdose of anesthesia, and the femur samples were taken out and fixed with 4% paraformaldehyde solution for one week, followed by CT examination and femur photos. Table 1 shows the specific data of new bone volume ratio and trabecular separation. Figure 5 A in the middle is a CT reconstruction image. Compared with the large number of vacancies in the blank group, PC, PCA3 and PMMA bone cements can successfully fill the defect sites. PMMA is difficult to degrade due to its biological inertness, and little tissue grows into it. It can be clearly seen that the PCA3 bone cement group has integrated with the bone tissue due to its good biocompatibility, but PMMA exists independently of the bone tissue due to its biological inertness. Figure 5 B in the figure is the new bone volume ratio. It can be seen that the amount of new bone formation in the PCA3 group is greater than that in the other two groups, up to 21%, and has a more excellent effect in promoting bone tissue regeneration. Figure 5 Middle C is the separation of trabeculae in each group. The PCA3 group has a lower separation than the other groups, which is 0.655 mm, indicating that the bone tissue promoted by this group is denser than that of other groups. In summary, the bone cement of the PCA3 group has the best repair-promoting effect among the above groups.

[0199] Table 1 New bone volume ratio and trabecular separation at the bone defect site 6 weeks after bone cement implantation

[0200]

[0201] As can be seen from the above results, the bone cement provides an effective supporting effect at the bone defect site, preventing the occurrence of osteonecrosis. At the same time, its good biocompatibility is also conducive to bone tissue regeneration, and ultimately a better bone repair effect than PMMA bone cement can be obtained.

[0202] In summary, the injectable and biodegradable high-strength porous bone cement of the present invention has good injectability, a long operation period, can quickly cure at 37°C, and does not generate heat during the curing process. At the same time, the cured bone cement has high strength and elasticity and does not disperse when encountering water. In addition, the rheological properties, mechanical properties and bone adhesion properties of the bone cement slurry can be regulated by adjusting the ratios of PEGS-OH, CPC and ALN. Among them, the sample with an ALN mass fraction of 3% has appropriate injectability and the highest modulus, as well as excellent bone adhesion and comprehensive osteogenic properties. Therefore, the bone cement of the present invention can be used for irregular bone defects in pathological conditions, playing a role of filling and supporting, and promoting bone regeneration through strong adhesion to bone tissue, and is an injectable bone cement material with clinical application prospects.

[0203] All documents mentioned in the present invention are incorporated herein by reference as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. An injectable bone cement composition, characterized in that: The injectable bone cement composition comprises: (c1) a liquid phase component, the liquid phase component comprising a polyester polymer; and (c2) a solid phase component, the solid phase component comprising a combination of phosphate and a phosphate derivative; The injectable bone cement composition further comprises a cross-linking agent; Wherein, the mass ratio of the liquid phase component to the solid phase component is 0.5:9.5-5:5; The phosphate derivative is selected from one or a mixture of two or more of etidronate sodium, clodronate sodium, pamidronate sodium, tiludronate sodium, alendronate sodium, neridronate sodium, olpadronate sodium, risedronate sodium, ibandronate sodium, and zoledronic acid; The mass fraction of the phosphate derivative is 0.001% to 10%; The polyester polymer is polyhydroxy polyethylene glycol polyglycerol sebacate PEGS-OH, Wherein, the structural unit of polyhydroxy polyethylene glycol polyglycerol sebacate PEGS-OH is as shown in Formula Ia or Formula Ib: Wherein, in Formula Ia, m1 is an integer of 1-20, and n1 is an integer of 5-60; In Formula Ib, m2 is an integer of 1-20, and n2 is an integer of 5-60.

2. The injectable bone cement composition according to claim 1, characterized in that The phosphate is selected from one of tetracalcium phosphate, monocalcium phosphate, tricalcium phosphate, or a mixture of two or more thereof.

3. The injectable bone cement composition according to claim 1, characterized in that The mass ratio of the polyester polymer to the cross-linking agent is 1:0.25-2.

4. The injectable bone cement composition according to claim 1, characterized in that The phosphate derivative is alendronate sodium.

5. The injectable bone cement composition according to claim 1, characterized in that The cross-linking agent is an isocyanate cross-linking agent.

6. The injectable bone cement composition according to claim 1, characterized in that The mass fraction of the phosphate derivative is 0.01%-5%.

7. The injectable bone cement composition according to claim 1, characterized in that The mass fraction of the phosphate derivative is 0.005%-5%.

8. An injectable bone cement, characterized in that: The injectable bone cement is obtained by mixing the liquid phase component and the solid phase component of the injectable bone cement composition according to any one of claims 1 to 7 and an optional cross-linking agent.

9. A kit for preparing the injectable bone cement according to claim 8, characterized in that: The kit comprises: (a) a first container, and a liquid component located in the container; (b) a second container, and a solid phase component located in the container; and optionally (c) a third container, and a cross-linking agent located within the container; In the first container, the liquid phase component exists in the form of a viscous liquid; in the second container, the solid phase component exists in the form of a powder; in the third container, the cross-linking agent exists in the form of a liquid. Wherein, the liquid phase component, solid phase component and cross-linking agent are the same as described above.

10. A use of the injectable bone cement as claimed in claim 8, characterized in that: For any one or two or more of the following applications: (i) preparing tissue defect filling materials; (ii) preparing materials for guiding tissue regeneration; (iii) preparing medical cosmetic materials; (iv) 3D printing matrix materials; (v) A loading matrix or carrier for growth factors or drugs.

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