Polymer artificial bone as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedical engineering, and particularly relates to a macromolecular artificial bone as well as a preparation method and application thereof. The invention provides a high-molecular artificial bone, preparation raw materials comprise a solvent and a solid raw material in a mass ratio of (80-90): (10-20), and the solid raw material comprises a high-molecular artificial bone matrix material, hydroxyapatite, metal phosphate or a raw material for preparing the metal phosphate, and I-type collagen / hyaluronic acid; the preparation raw materials also comprise a pore-forming agent. The high-molecular artificial bone provided by the invention has high biocompatibility, high porosity, super-hydrophilic property, sufficient mechanical property and degradability, and meanwhile, the artificial bone scaffold is compounded with metal ions which have a promoting effect on osteogenesis, so that bone regeneration can be promoted by releasing the metal ions, and the bone regeneration rate is increased. The defects of an existing clinical artificial bone material in the aspects of biocompatibility and osteoinductivity are overcome.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering technology, specifically relating to a polymeric artificial bone, its preparation method, and its application. Background Technology

[0002] Currently, clinically used artificial bone materials mainly include metals (such as titanium alloys), bioceramics (such as hydroxyapatite and β-tricalcium phosphate), and polymers (such as PLGA and PCL). However, these materials still have significant shortcomings in terms of biocompatibility, mechanical property matching, osteoinductive properties, and controllable degradation. While metal materials (such as titanium alloys) possess excellent mechanical strength, they lack bioactivity, and long-term implantation may lead to stress shielding effects, hindering normal bone remodeling. Furthermore, the non-degradability of metal materials necessitates a second surgery for removal after bone healing, increasing the burden on patients. Bioceramic materials (such as hydroxyapatite), while exhibiting good osteoconductivity, are brittle and lack toughness, making them prone to fracture under stress. Additionally, ceramic materials degrade slowly, and the degradation process may cause local pH changes, affecting the growth environment of surrounding cells. Polymers (such as PLGA and PCL) are degradable and have good processing properties, but their mechanical strength is generally low, making it difficult to meet the needs of load-bearing bone repair. Moreover, pure polymers lack osteoinductive activity and cannot actively promote bone regeneration.

[0003] In recent years, researchers have attempted to improve the performance of artificial bone through composite modification. For example, they have used pore-forming agents (such as NaCl) to construct porous structures. However, scaffolds prepared by traditional leaching methods exhibit uneven pore distribution and poor hydrophilicity, affecting early cell adhesion. Combining polymers with ceramics (such as PLGA / hydroxyapatite) to optimize mechanical properties has also been explored, but the interfacial bonding between the two phases is weak, making delamination likely. Therefore, existing artificial bone materials still struggle to simultaneously meet the requirements of high biocompatibility, controllable degradation, mechanical adaptability, high porosity, and osteogenic induction activity.

[0004] Therefore, providing an artificial bone material that simultaneously meets the requirements of high biocompatibility, controllable degradation, mechanical compatibility, high porosity, and osteogenic induction activity has become a problem that needs to be solved. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a method for preparing polymeric artificial bone and its application. The polymeric artificial bone provided by the present invention can simultaneously meet the requirements of high biocompatibility, controllable degradation, mechanical adaptation, high porosity and osteogenic induction activity.

[0006] This invention provides a polymeric artificial bone, the raw materials for which are prepared include a solvent and a solid raw material in a mass ratio of 80-90:10-20, wherein the solid raw material includes:

[0007] 70-90 parts by weight of polymeric artificial bone matrix material;

[0008] 5-20 parts by weight of hydroxyapatite;

[0009] 5-10 parts by weight of metal phosphate, or 0-10 parts by weight of raw materials for preparing metal phosphate;

[0010] 1-5 parts by weight of type I collagen / hyaluronic acid;

[0011] The raw materials also include a pore-forming agent, the volume of which accounts for 80%-95% of the total volume of the pore-forming agent, the polymeric artificial bone matrix material, and the hydroxyapatite.

[0012] Preferably, the polymeric artificial bone matrix material is selected from at least one of polylactic acid-glycolic acid copolymer, polylactic acid, and polycaprolactone.

[0013] Preferably, the metal element in the metal phosphate is selected from at least one of Mg, Zn, Sr, Mn, Co, and Cu;

[0014] The raw materials for preparing metal phosphates include soluble metal salts and soluble phosphates. The soluble metal salts are selected from chloride salts of Mg, Zn, Sr, Mn, Co, and Cu metals, and the soluble phosphates are sodium or potassium salts containing hydrogen phosphate or dihydrogen phosphate.

[0015] Preferably, in the type I collagen / hyaluronic acid, the type I collagen has a molecular weight of 100~300kDa, and the hyaluronic acid has a molecular weight of 800~1000kDa.

[0016] Preferably, the pore-forming agent is selected from sodium chloride and sucrose;

[0017] The particle size of the pore-forming agent is 200~400μm.

[0018] Preferably, the solvent is selected from at least one of dichloromethane, trichloromethane, and N,N-dimethylformamide.

[0019] Preferably, the porosity of the polymeric artificial bone is 80%-95%, and the pore size is 200-400μm.

[0020] This invention also provides a method for preparing the above-mentioned polymeric artificial bone, which can be any one of method one to method three.

[0021] Method 1 includes the following steps:

[0022] A) Mix type I collagen / hyaluronic acid, hydroxyapatite, metal phosphate, polymeric artificial bone matrix material and solvent to obtain a mixture;

[0023] B) Mix the mixture with the pore-forming agent to obtain a mixture;

[0024] C) After the mixture is shaped, the pore-forming agent is removed, and the mixture is freeze-dried to obtain biocompatible polymeric artificial bone.

[0025] Preferably, step A) includes:

[0026] A1) Type I collagen / hyaluronic acid, hydroxyapatite, and metal phosphates are added to a mixed solvent and dispersed to obtain a mixed solution;

[0027] A2) The polymeric artificial bone matrix material is mixed with the mixed solution to obtain a mixed solution.

[0028] Method 2 includes the following steps:

[0029] A) A mixture is prepared by mixing hydroxyapatite, metal phosphate, polymeric artificial bone matrix material, and solvent;

[0030] B) Mix the mixture with the pore-forming agent to obtain a mixture;

[0031] C) After the mixture is shaped, the pore-forming agent is removed, and the mixture is freeze-dried to obtain polymeric artificial bone.

[0032] D) Dissolve type I collagen / hyaluronic acid in water once, immerse the artificial bone in the type I collagen / hyaluronic acid aqueous solution, and freeze-dry to obtain biocompatible polymeric artificial bone.

[0033] Preferably, step A) includes:

[0034] A1) Hydroxyapatite and metal phosphates are added to a mixed solvent and dispersed to obtain a mixed solution;

[0035] A2) The polymeric artificial bone matrix material is mixed with the mixed solution to obtain a mixed solution.

[0036] Method 3 includes the following steps:

[0037] A) A mixture is prepared by mixing hydroxyapatite, polymeric artificial bone matrix material and solvent;

[0038] B) Mix the mixture with the pore-forming agent to obtain a mixture;

[0039] C) After the mixture is allowed to stand and solidify, the pore-forming agent is removed, and the mixture is freeze-dried to obtain polymeric artificial bone.

[0040] D) Dissolve type I collagen / hyaluronic acid, soluble metal salt, and soluble phosphate in water once to obtain a mixed solution;

[0041] E) Immerse the artificial bone in the mixed solution, then freeze-dry to obtain biocompatible polymeric artificial bone.

[0042] Preferably, step D) includes:

[0043] 1) Dissolve type I collagen / hyaluronic acid in water once to obtain solution A;

[0044] 2) Dissolve the soluble metal salt in solution A to obtain solution B;

[0045] 3) Dissolve the soluble phosphate in solution A to obtain solution C;

[0046] 4) Mix solution B and solution C to obtain a mixed solution.

[0047] The present invention also provides an application of the above-mentioned polymeric artificial bone in the preparation of tissue engineering scaffolds and drug sustained-release carriers.

[0048] Compared with existing technologies, a polymeric artificial bone is prepared by using a solvent and solid raw materials in a mass ratio of 80-90:10-20. The solid raw materials include: 70-90 parts by mass of polymeric artificial bone matrix material; 5-20 parts by mass of hydroxyapatite; 0-10 parts by mass of metal phosphate, or 0-10 parts by mass of raw materials for preparing metal phosphate; and 0-5 parts by mass of type I collagen / hyaluronic acid. The preparation also includes a pore-forming agent, the volume of which accounts for 80%-95% of the total volume of the pore-forming agent, polymeric artificial bone matrix material, and hydroxyapatite. The polymeric artificial bone provided by this invention possesses high biocompatibility, high porosity, superhydrophilic properties, sufficient mechanical properties, and biodegradability. Furthermore, this artificial bone scaffold incorporates metal ions that promote osteogenic formation, which can promote bone regeneration by releasing these metal ions, thus overcoming the shortcomings of existing clinical artificial bone materials in terms of biocompatibility and osteoinductive properties. Attached Figure Description

[0049] Figure 1 Scanning electron microscope (SEM) images and energy scattering spectra (EDS) images of different polymeric artificial bone scaffolds prepared for the examples;

[0050] Figure 2 Porosity characterization of different polymeric artificial bone scaffolds prepared for the examples;

[0051] Figure 3 The compressive strength test results of different polymeric artificial bone scaffolds prepared for the examples;

[0052] Figure 4 Dynamic water contact angle test results of different polymeric artificial bone scaffolds prepared for the examples;

[0053] Figure 5Degradation curves of the PLGA / Co scaffold;

[0054] Figure 6 Scanning electron microscope (SEM) images and energy dispersive spectroscopy (EDS) images of different polymeric artificial bone scaffolds PLGA / Co, PLGA / Mg, PLGA / Mn, and PLGA / Zn prepared for the examples;

[0055] Figure 7 Porosity characterization of different polymeric artificial bone scaffolds prepared for the examples;

[0056] Figure 8 The compressive strength test results of different polymeric artificial bone scaffolds prepared for the examples;

[0057] Figure 9 Dynamic water contact angle test results of different polymeric artificial bone scaffolds prepared for the examples;

[0058] Figure 10 Scanning electron microscopy (SEM) images and energy dispersive spectroscopy (EDS) images of different polymeric artificial bone scaffolds PLGA / Co and PLGA / Mg prepared for the examples;

[0059] Figure 11 Porosity characterization of different polymeric artificial bone scaffolds prepared for the examples;

[0060] Figure 12 The compressive strength test results of different polymeric artificial bone scaffolds prepared for the examples;

[0061] Figure 13 Dynamic water contact angle test results of different polymeric artificial bone scaffolds prepared for the examples;

[0062] Figure 14 The results of dynamic water contact angle test for the polymer artificial bone scaffold prepared in Comparative Example 1;

[0063] Figure 15 Calcein-AM / PI staining results of bone marrow mesenchymal stem cells (NUSCs) from the affected area of ​​atrophic nonunion after 3 days of culture on two types of three-dimensional porous composite scaffolds, PLGA / nHA and PLGA / Co.

[0064] Figure 16 The results of the effects of different polymeric artificial bone scaffolds prepared for the examples on NUSC cell adhesion. Detailed Implementation

[0065] This invention provides a polymeric artificial bone, the raw materials for which are prepared include a solvent and a solid raw material in a mass ratio of 80-90:10-20, wherein the solid raw material includes:

[0066] 70-90 parts by weight of polymeric artificial bone matrix material;

[0067] 5-20 parts by weight of hydroxyapatite;

[0068] 0-10 parts by weight of metal phosphate, or 0-10 parts by weight of raw materials for preparing metal phosphate;

[0069] 0-5 parts by weight of type I collagen / hyaluronic acid;

[0070] It also includes a pore-forming agent, the volume of which accounts for 80-95% of the total volume of the pore-forming agent, the polymeric artificial bone matrix material, and the hydroxyapatite.

[0071] The raw materials for preparing the polymeric artificial bone include solvents and solid raw materials. The mass ratio of the solvent to the solid raw materials is 80~90:10~20, and can be any ratio between 80:20, 81:19, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, or 80~90:10~20.

[0072] The solvent is selected from one or more of dichloromethane, trichloromethane, and N,N-dimethylformamide (DMF). Preferably, the solvent is a mixed solution of dichloromethane and N,N-dimethylformamide in a volume ratio of 2:1. The solution selected in this invention can effectively dissolve the polymer matrix material and induce phase separation through solvent evaporation during scaffold fabrication, forming a microporous structure on the pore wall surface and improving pore connectivity.

[0073] The solid raw material for the polymeric artificial bone provided by this invention comprises 70-90 parts by weight of a polymeric artificial bone matrix material, which can be 70, 75, 80, 85, 90, or any value between 70 and 90 parts by weight. The polymeric artificial bone matrix material is selected from one or more of polylactic acid-glycolic acid copolymer (PLGA), polylactic acid (PLA), and polycaprolactone (PCL). Preferably, it is polylactic acid-glycolic acid copolymer (PLGA).

[0074] The solid raw material of the polymeric artificial bone provided by the present invention further includes 5 to 20 parts by weight of hydroxyapatite, which can be 5, 7, 10, 12, 15, 17, 20, or any value between 5 and 20 parts by weight.

[0075] The solid raw material for polymeric artificial bone provided by this invention further includes 0-10 parts by mass of a metal phosphate, which can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any value between 0 and 10 parts by mass. The metal element in the metal phosphate is selected from at least one of Mg, Zn, Sr, Mn, Co, and Cu. Alternatively, the solid raw material for polymeric artificial bone provided by this invention further includes 0-10 parts by mass of a raw material for preparing the metal phosphate, which can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any value between 0 and 10 parts by mass. The raw material for preparing the metal phosphate includes a soluble metal salt and a soluble phosphate. The soluble metal salt is selected from chloride salts of Mg, Zn, Sr, Mn, Co, and Cu metals. The soluble phosphate is a sodium or potassium salt containing hydrogen phosphate or dihydrogen phosphate. Further, the soluble phosphate is one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate. The ions contained in both soluble metal salts and soluble phosphates mineralize in solution, resulting in the final scaffold containing metal phosphates. The bioactive ions in the metal phosphates can achieve stable loading and sustained release within the polymeric artificial bone, continuously promoting bone regeneration.

[0076] Preferably, the metal element in the metal phosphate is selected from Co. Compared with other elements, Co has a unique hypoxia-simulating effect, which can activate the HIF-1α signaling pathway at low doses, synergistically promote angiogenesis and osteogenic differentiation, accelerate early blood revascularization, and significantly improve the bone defect repair effect.

[0077] The solid raw material of the polymeric artificial bone provided by this invention further includes 0-5 parts by weight of type I collagen / hyaluronic acid, which can be 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, or any value between 0 and 5 parts by weight. In the type I collagen / hyaluronic acid, the molecular weight of type I collagen is 100-300 kDa, which can be 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, or any value between 100 and 300 kDa; the molecular weight of hyaluronic acid is 800-1000 kDa, which can be 800 kDa, 850 kDa, 900 kDa, 950 kDa, 1000 kDa, or any value between 800 and 1000 kDa. Type I collagen provides natural sites for cell adhesion recognition, promoting osteoblast migration and spreading; hyaluronic acid maintains a local moist microenvironment through its hydrophilic network, regulating cell behavior; both can mimic the organic components in the natural bone matrix, enhancing the integration of materials with host tissues and accelerating the bone defect repair process.

[0078] The raw materials for preparing polymeric artificial bone provided by this invention also include a pore-forming agent, wherein the volume of the pore-forming agent accounts for 80% to 95% of the total volume of the pore-forming agent, the polymeric artificial bone matrix material, and hydroxyapatite. The pore-forming agent is selected from sodium chloride and sucrose; the particle size of the pore-forming agent is 200 to 400 μm.

[0079] The polymeric artificial bone provided by this invention has a porosity of 80%~95% and a pore size of 200~400μm. The polymeric artificial bone possesses a highly porous (>80%) and interconnected porous structure, which facilitates cell migration and nutrient exchange; furthermore, it exhibits superhydrophilic surface properties, enhancing cell adhesion and early osteogenic formation.

[0080] This invention also provides three methods for preparing the above-mentioned polymeric artificial bone.

[0081] Method 1 includes the following steps:

[0082] A) Mix type I collagen / hyaluronic acid, hydroxyapatite, metal phosphate, polymeric artificial bone matrix material and solvent to obtain a mixture;

[0083] B) Mix the mixture with the pore-forming agent to obtain a mixture;

[0084] C) After the mixture is shaped, the pore-forming agent is removed, and the mixture is freeze-dried to obtain biocompatible polymeric artificial bone.

[0085] This invention first prepares a mixture, including the following steps:

[0086] A1) Type I collagen / hyaluronic acid, hydroxyapatite, and metal phosphates are added to a mixed solvent and dispersed to obtain a mixed solution;

[0087] A2) The polymeric artificial bone matrix material is mixed with the mixed solution to obtain a mixed solution.

[0088] Specifically, the present invention first dries and removes water from hydroxyapatite, metal phosphate and pore-forming agent.

[0089] Type I collagen / hyaluronic acid, hydroxyapatite, and metal phosphates are added to the mixed solvent and dispersed. This invention does not impose any particular limitations on the mixing method; ultrasonic dispersion is acceptable. Then, polymeric artificial bone matrix material is added and stirred until completely dissolved to obtain a mixture.

[0090] After obtaining the mixture, it is mixed with a pore-forming agent to obtain a final mixture. Then, the mixture is poured into a mold, allowed to stand and solidify, and the pore-forming agent is removed. In this invention, a single water immersion is preferably used to remove the pore-forming agent. Finally, freeze-drying is performed to obtain biocompatible polymeric artificial bone.

[0091] Method 2 includes the following steps:

[0092] a) A mixture is prepared by mixing hydroxyapatite, metal phosphate, polymeric artificial bone matrix material, and solvent;

[0093] b) Mix the mixture with the pore-forming agent to obtain a mixture;

[0094] c) After the mixture is allowed to stand and solidify, it is soaked in water to remove the pore-forming agent, and then freeze-dried to obtain polymeric artificial bone.

[0095] d) Dissolve type I collagen / hyaluronic acid in water once, immerse the artificial bone in the type I collagen / hyaluronic acid aqueous solution, freeze dry, and obtain biocompatible polymeric artificial bone.

[0096] Specifically, the present invention first dries and removes water from hydroxyapatite, metal phosphate and pore-forming agent.

[0097] Hydroxyapatite and metal phosphates are added to the mixed solvent and dispersed. This invention does not impose any particular limitation on the mixing method; ultrasonic dispersion is acceptable. Then, polymeric artificial bone matrix material is added and stirred until completely dissolved to obtain a mixed solution.

[0098] After obtaining the mixture, it is mixed with a pore-forming agent to obtain a final mixture. Then, the mixture is poured into a mold, allowed to stand and solidify, and the pore-forming agent is removed. In this invention, a single water immersion is preferably used to remove the pore-forming agent. The mixture is then freeze-dried to obtain polymeric artificial bone.

[0099] Type I collagen / hyaluronic acid was dissolved in water once, and the polymeric artificial bone was then immersed in the type I collagen / hyaluronic acid aqueous solution. Finally, freeze-drying was performed to obtain biocompatible polymeric artificial bone.

[0100] Method 3 includes the following steps:

[0101] 1) A mixture is prepared by mixing hydroxyapatite, polymeric artificial bone matrix material, and solvent;

[0102] 2) The mixture is mixed with a pore-forming agent to obtain a mixture;

[0103] 3) After the mixture is allowed to stand and solidify, it is soaked in water to remove the pore-forming agent, and then freeze-dried to obtain polymeric artificial bone.

[0104] 4) Dissolve type I collagen / hyaluronic acid, soluble metal salt, and soluble phosphate in water once to obtain a mixed solution;

[0105] 5) Immerse the artificial bone in the mixed solution and freeze-dry to obtain polymeric artificial bone.

[0106] Specifically, the present invention first dries and removes water from hydroxyapatite and pore-forming agent.

[0107] Hydroxyapatite is added to the mixed solvent and dispersed. This invention does not impose any particular limitation on the mixing method; ultrasonic dispersion is acceptable. Then, polymeric artificial bone matrix material is added and stirred until completely dissolved to obtain a mixture.

[0108] After obtaining the mixture, it is mixed with a pore-forming agent to obtain a final mixture. Then, the mixture is poured into a mold, allowed to stand and solidify, and the pore-forming agent is removed. In this invention, a single water immersion is preferably used to remove the pore-forming agent. The mixture is then freeze-dried to obtain polymeric artificial bone.

[0109] Type I collagen / hyaluronic acid is dissolved in water once to obtain solution A; a soluble metal salt is dissolved in solution A to obtain solution B; a soluble phosphate is dissolved in solution A to obtain solution C; solutions B and C are mixed to obtain mixed solution D.

[0110] The polymeric artificial bone was immersed in mixed solution D. Finally, it was freeze-dried to obtain biocompatible polymeric artificial bone.

[0111] In this invention, the porous scaffold prepared by particle leaching combined with phase transformation has high porosity and pore connectivity; the main material of the scaffold is a polymer matrix, and the appropriate molecular weight enables the scaffold to have sufficient mechanical properties and a degradation rate that matches bone formation; the type I collagen / hyaluronic acid composite in the scaffold endows the scaffold with high biocompatibility and superhydrophilic properties.

[0112] The present invention also provides an application of the above-mentioned polymeric artificial bone in the preparation of tissue engineering scaffolds and drug sustained-release carriers.

[0113] The polymeric artificial bone provided by this invention has high biocompatibility, high porosity, superhydrophilic properties, sufficient mechanical properties and biodegradability. At the same time, the artificial bone scaffold is compounded with metal ions that promote osteogenic formation, and can promote bone regeneration by releasing metal ions.

[0114] To further understand the present invention, the preparation method and application of the polymeric artificial bone provided by the present invention will be described below with reference to the embodiments. The scope of protection of the present invention is not limited by the following embodiments.

[0115] Type I collagen / hyaluronic acid was purchased from Beijing Solarbio Technology Co., Ltd.

[0116] PLGA is synthesized from glycolide and lactide via ring-opening polymerization, and its weight-average molecular weight M w It has a molecular weight distribution coefficient of 100 kDa and a molecular weight distribution coefficient of less than 1.5, with an LA:GA ratio of 75:25.

[0117] Example 1

[0118] 1. Preparation of different polymeric artificial bone scaffolds

[0119] (1) Dry hydroxyapatite and different types of metal phosphates M3(PO4)2 (Co3(PO4)2, Mg3(PO4)2, Sr3(PO4)2 or Zn3(PO4)2) to remove water;

[0120] (2) Screen out sodium chloride with a particle size of 200-400 micrometers and dry it to remove water;

[0121] (3) Add 0.5g of type I collagen / hyaluronic acid, 0.5g of hydroxyapatite, and 0.5g of different types of M3(PO4)2 to a mixed solvent of 38 ml of dichloromethane and 19 ml of N,N-dimethylformamide (DMF), and sonicate for 15 minutes to disperse it evenly to obtain solution A;

[0122] (4) Weigh 9 g of PLGA, the matrix material of polymeric artificial bone, add it to the mixed solution, and stir until completely dissolved;

[0123] (5) Add 101.4 g of sodium chloride and stir to obtain a homogeneous mixture. Pour the mixture into a mold (40×40×10mm). 3 After standing for 24 hours to form, the material is removed, soaked in water for 3 days to remove the pore-forming agent, and then freeze-dried to obtain polymeric artificial bone, which is denoted as PLGA / Co, PLGA / Mg, PLGA / Sr, and PLGA / Zn, respectively.

[0124] 2. Characterization of the different polymeric artificial bones prepared above.

[0125] (1) Scanning electron microscopy (SEM) was used to characterize the porous structure of the scaffold: the scaffold was immersed in liquid nitrogen and frozen until it fractured. The fracture surface was marked and freeze-dried. Then, it was sputtered with gold and observed using a field emission scanning electron microscope.

[0126] (2) Energy scattering spectroscopy (EDS) characterizes the elemental composition of the scaffold: the scaffold is immersed in liquid nitrogen and frozen until it is brittle. The fracture surface is marked and freeze-dried. Then, gold is sputtered and EDS scan is performed.

[0127] See results Figure 1 , Figure 1 Scanning electron microscopy (SEM) images and energy dispersive spectroscopy (EDS) images of different polymeric artificial bone scaffolds PLGA / Co, PLGA / Mg, PLGA / Sr, and PLGA / Zn prepared for the examples.

[0128] (3) Porosity testing characterized the high porosity of the artificial bone scaffold: a size of 40×40×10mm was used. 3 The porosity of the support was measured using a true density analyzer, and the measurement was repeated three times.

[0129] See results Figure 2 , Figure 2 Porosity characterization of different polymeric artificial bone scaffolds prepared for the examples.

[0130] (4) Compression strength test: Each group of porous materials is made into a 10 mm × 10 mm × 4 mm specimen. According to the national standard GB / T1041-2008, the prepared specimen is placed in the test area of ​​the testing machine and loaded at a speed of 5 mm / min to carry out the compression test until the specimen is deformed, and the maximum compression strength is recorded.

[0131] See results Figure 3 , Figure 3 The compressive strength test results of different polymeric artificial bone scaffolds prepared for the examples are shown. Figure 3 It can be seen that PLGA / Co is 3.4 MPa, PLGA / Mg is 3.54 MPa, PLGA / Sr is 3.47 MPa, and PLGA / Zn is 3.46 MPa.

[0132] (5) Water contact angle test characterizes the superhydrophilic properties of the stent: a flat stent surface is selected as the test surface, and a dynamic water contact angle meter is used to characterize the hydrophilicity.

[0133] See results Figure 4 , Figure 4 The results of dynamic water contact angle tests on different polymeric artificial bone scaffolds prepared for the examples are shown. Figure 4 It can be seen that water droplets dripping onto the surface of the support can be completely absorbed into the pores by the support within 2 seconds.

[0134] (6) Degradation test: The degradation performance of the porous scaffold was evaluated using an in vitro simulated degradation method. Precisely weighed (W0) scaffold samples (10mm × 10mm × 2mm) were immersed in 50mL of phosphate-buffered saline (PBS, pH=7.4) and incubated continuously in a 37℃ constant temperature shaking incubator (100rpm). Samples were periodically removed (on days 1, 3, 7, 14, 21, and 28), rinsed with deionized water, and vacuum dried to constant weight (W1). The mass loss rate was calculated (Degradation% = (W0 - W1) / W0 × 100%).

[0135] See results Figure 5 , Figure 5 Degradation curves for the PLGA / Co scaffold. (From...) Figure 5It can be seen that the scaffold begins to degrade after 3 weeks in an in vitro simulated environment, and its mass degrades to less than 50% after 8 weeks. Its degradation rate can match the rate of new bone formation.

[0136] Example 2

[0137] 1. Preparation of different polymeric artificial bone scaffolds

[0138] (1) Dry hydroxyapatite and different types of metal phosphates M3(PO4)2 (Co3(PO4)2, Mg3(PO4)2, Mn3(PO4)2 or Zn3(PO4)2) to remove water;

[0139] (2) Screen out sodium chloride with a particle size of 200-400 micrometers and dry it to remove water;

[0140] (3) Add 0.5 g of hydroxyapatite and 0.5 g of different types of M3(PO4)2 to a mixed solvent of 38 ml of dichloromethane and 19 ml of N,N-dimethylformamide (DMF), and sonicate for 15 minutes to disperse it evenly to obtain solution A;

[0141] (4) Weigh 9 g of PLGA, the matrix material of polymeric artificial bone, add it to mixed solution A, and stir until completely dissolved;

[0142] (5) Add 101.4 g of sodium chloride and stir to obtain a homogeneous mixture. Pour the mixture into a mold (40×40×10mm). 3 After standing for 24 hours to form, the material is removed, soaked in water for 3 days to remove the pore-forming agent, and then freeze-dried to obtain polymeric artificial bone.

[0143] (6) Dissolve 1g of type I collagen completely in 20ml of deionized water to obtain solution B; immerse the above polymeric artificial bone in solution B, take it out and rinse it with water once, freeze dry it to obtain biocompatible polymeric artificial bone, which are respectively PLGA / Co, PLGA / Mg, PLGA / Mn and PLGA / Zn.

[0144] 2. Characterization of the different polymeric artificial bones prepared above.

[0145] (1) Scanning electron microscopy (SEM) was used to characterize the porous structure of the scaffold: the scaffold was immersed in liquid nitrogen and frozen until it fractured. The fracture surface was marked and freeze-dried. Then, it was sputtered with gold and observed using a field emission scanning electron microscope.

[0146] (2) Energy scattering spectroscopy (EDS) characterizes the elemental composition of the scaffold: the scaffold is immersed in liquid nitrogen and frozen until it is brittle. The fracture surface is marked and freeze-dried. Then, gold is sputtered and EDS scan is performed.

[0147] See results Figure 6 , Figure 6 Scanning electron microscope (SEM) images and energy dispersive spectroscopy (EDS) images of different polymeric artificial bone scaffolds PLGA / Co, PLGA / Mg, PLGA / Mn, and PLGA / Zn prepared for the examples.

[0148] (3) Porosity testing characterized the high porosity of the artificial bone scaffold: a size of 40×40×10mm was used. 3 The porosity of the support was measured using a true density analyzer, and the measurement was repeated three times.

[0149] See results Figure 7 , Figure 7 Porosity characterization of different polymeric artificial bone scaffolds prepared for the examples.

[0150] (4) Compression strength test: Each group of porous materials is made into a 10 mm × 10 mm × 4 mm specimen. According to the national standard GB / T1041-2008, the prepared specimen is placed in the test area of ​​the testing machine and loaded at a speed of 5 mm / min to carry out the compression test until the specimen is deformed, and the maximum compression strength is recorded.

[0151] See results Figure 8 , Figure 8 The compressive strength test results of different polymeric artificial bone scaffolds prepared for the examples are shown. Figure 8 It can be seen that PLGA / Co is 3.58 MPa, PLGA / Mg is 3.55 MPa, PLGA / Mn is 3.45 MPa, and PLGA / Zn is 3.50 MPa.

[0152] (5) Water contact angle test characterizes the superhydrophilic properties of the stent: a flat stent surface is selected as the test surface, and a dynamic water contact angle meter is used to characterize the hydrophilicity.

[0153] See results Figure 9 , Figure 9 The results of dynamic water contact angle tests on different polymeric artificial bone scaffolds prepared for the examples are shown. Figure 9 It can be seen that water droplets dripping onto the surface of the support can be completely absorbed into the pores by the support within 2 seconds.

[0154] Example 3

[0155] 1. Preparation of different polymeric artificial bone scaffolds

[0156] (1) Dry hydroxyapatite to remove water;

[0157] (2) Screen out sodium chloride with a particle size of 200-400 micrometers and dry it to remove water;

[0158] (3) Add 1 g of hydroxyapatite to a mixed solvent of 38 ml dichloromethane and 19 ml N,N-dimethylformamide (DMF), and sonicate for 15 minutes to disperse it evenly to obtain solution A;

[0159] (4) Weigh 9 g of PLGA, the matrix material of polymeric artificial bone, add it to mixed solution A, and stir until completely dissolved;

[0160] (5) Add 101.4 g of sodium chloride and stir to obtain a homogeneous mixture. Pour the mixture into a mold (40×40×10mm). 3 After standing for 24 hours to form, the material is removed, soaked in water for 3 days to remove the pore-forming agent, and then freeze-dried to obtain polymeric artificial bone.

[0161] (6) Dissolve 1g of type I collagen completely in 20ml of deionized water to obtain solution B;

[0162] (7) Dissolve different kinds of soluble metal salts (3.57 mg CoCl2·6H2O, 3.05 mg MgCl2) in 5 ml of solution B to obtain solution C; dissolve 0.672 g NaH2PO4 in 5 ml of solution B to obtain solution D; mix solution C and solution D to obtain solution E.

[0163] (8) The above-mentioned polymeric artificial bone was immersed in solution E, taken out and rinsed with water once, and freeze-dried to obtain biocompatible polymeric artificial bone, which was denoted as PLGA / Co and PLGA / Mg respectively.

[0164] 2. Characterization of the different polymeric artificial bones prepared above.

[0165] (1) Scanning electron microscopy (SEM) was used to characterize the porous structure of the scaffold: the scaffold was immersed in liquid nitrogen and frozen until it fractured. The fracture surface was marked and freeze-dried. Then, it was sputtered with gold and observed using a field emission scanning electron microscope.

[0166] (2) Energy scattering spectroscopy (EDS) characterizes the elemental composition of the scaffold: the scaffold is immersed in liquid nitrogen and frozen until it is brittle. The fracture surface is marked and freeze-dried. Then, gold is sputtered and EDS scan is performed.

[0167] See results Figure 10 , Figure 10 Scanning electron microscopy (SEM) images and energy dispersive spectroscopy (EDS) images of different polymeric artificial bone scaffolds PLGA / Co and PLGA / Mg prepared for the examples.

[0168] (3) Porosity testing characterized the high porosity of the artificial bone scaffold: a size of 40×40×10mm was used. 3The porosity of the support was measured using a true density analyzer, and the measurement was repeated three times.

[0169] See results Figure 11 , Figure 11 Porosity characterization of different polymeric artificial bone scaffolds prepared for the examples.

[0170] (4) Compression strength test: Each group of porous materials is made into a 10 mm × 10 mm × 4 mm specimen. According to the national standard GB / T1041-2008, the prepared specimen is placed in the test area of ​​the testing machine and loaded at a speed of 5 mm / min to carry out the compression test until the specimen is deformed, and the maximum compression strength is recorded.

[0171] See results Figure 12 , Figure 12 The compressive strength test results of different polymeric artificial bone scaffolds prepared for the examples are shown. Figure 12 It can be seen that the PLGA / Co ratio is 3.63 MPa and the PLGA / Mg ratio is 3.59 MPa.

[0172] (5) Water contact angle test characterizes the superhydrophilic properties of the stent: a flat stent surface is selected as the test surface, and a dynamic water contact angle meter is used to characterize the hydrophilicity.

[0173] See results Figure 13 , Figure 13 The results of dynamic water contact angle tests on different polymeric artificial bone scaffolds prepared for the examples are shown. Figure 13 It can be seen that water droplets dripping onto the surfaces of both types of supports can be completely absorbed into the pores of the supports within 2 seconds.

[0174] Comparative Example 1

[0175] Preparation of PLGA / nHA

[0176] (1) Dry hydroxyapatite to remove water;

[0177] (2) Screen out sodium chloride with a particle size of 200-400 micrometers and dry it to remove water;

[0178] (3) The mixed solution of 38 ml dichloromethane and 19 ml N,N-dimethylformamide (DMF) is denoted as solution A;

[0179] (4) Add 1 g of hydroxyapatite to solution A and sonicate for 15 minutes to disperse it evenly. Weigh 9 g of PLGA, the matrix material of polymeric artificial bone, and add it to the mixed solution. Stir until completely dissolved.

[0180] (5) Add 101.4g of pore-forming agent and stir to obtain a uniform mixture. Pour the mixture into a mold (40×40×10mm).3 After standing for 24 hours to form, the material is removed, soaked in water for 3 days to remove the pore-forming agent, and then freeze-dried to obtain a polymeric artificial bone, denoted as PLGA / nHA.

[0181] Test case

[0182] 1. Verification of the hydrophilicity of the polymeric artificial bone scaffold prepared in Comparative Example 1

[0183] A flat support surface was selected as the test surface, and hydrophilicity was characterized using a dynamic water contact angle meter. See the results below. Figure 14 , Figure 14 The results show the dynamic water contact angle of the polymer artificial bone scaffold prepared in Comparative Example 1. Figure 14 It can be seen that the water droplets dripped onto the surface of the PLGA / nHA scaffold were hardly absorbed into the pores of the scaffold within 2 seconds, indicating that the surface of the PLGA / nHA scaffold has hydrophobic properties.

[0184] 2. Verification of cell compatibility of the cobalt polymer artificial bone scaffold prepared in Example 3

[0185] The high cell compatibility of the artificial bone was verified by Calcein / PI staining, and the good cell adhesion of the scaffold was verified by SEM and fluorescence staining. The scaffold (8 mm in diameter and 3 mm in height) was placed in a 24-well plate, and BMSCs were cultured at a density of 1 × 10⁻⁶ cells / well. 5 After being seeded on the scaffold at high density and cultured for 1 and 3 days, staining was performed according to the following steps: (1) Calcein-AM and PI dyes were diluted 10 times with dye dilution buffer. Then, 40 μL of diluted Calcein-AM dye solution was added to 4 ml of serum-free medium to prepare Calcein-AM working solution, and 12 μL of diluted PI dye solution was added to 4 ml of serum-free medium to prepare PI working solution; (2) The well plate was centrifuged at 20 ℃ and 1200 rpm for 5 min. The medium was carefully removed, and the plate was washed twice with PBS. After each washing, the supernatant was carefully removed by centrifugation; (3) 200 μL of Calcein-AM working solution was added to each well and incubated at room temperature in the dark for 15 min. After centrifugation in the dark, the dye solution was removed, and the plate was washed twice with PBS in the same way; (4) 200 μL of PI working solution was added to each well and incubated at room temperature in the dark for 2 min. After centrifugation in the dark, the dye solution was removed, and the plate was washed twice with PBS in the same way; (5) The staining results of each group were observed under a fluorescence microscope, and the images were collected.

[0186] See results Figure 15 , Figure 15Calcein-AM / PI staining results of bone marrow mesenchymal stem cells (NUSCs) from the affected area of ​​atrophic nonunion after 3 days of culture on two types of three-dimensional porous composite scaffolds, PLGA / nHA and PLGA / Co.

[0187] Depend on Figure 15 It can be seen that when cells are seeded onto the PLGA / Co scaffold for culture, a large number of live cells and very few dead cells can be observed, indicating that the scaffold has good cell compatibility.

[0188] See results Figure 16 , Figure 16 The results of the effects of different polymeric artificial bone scaffolds prepared for the examples on NUSC cell adhesion. Figure 16 In the image, (a) shows the immunofluorescence staining of F-actin protein on NUSCs after culturing them for one day on two types of three-dimensional porous composite scaffolds, PLGA / nHA and PLGA / Co; (b) shows the morphology of the NUSCs on the two types of scaffolds observed by scanning electron microscopy.

[0189] Depend on Figure 16 It can be seen that on the PLGA / Co scaffold well wall, the cells are fully spread out in a typical spindle shape, with abundant pseudopodia that are tightly attached to the well wall surface, indicating that the scaffold has good cell adhesion. In contrast, the cell adhesion effect of PLGA / nHA scaffold is relatively poor due to the hydrophobic surface of the scaffold.

[0190] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A polymeric artificial bone, characterized in that, The preparation raw materials include a solvent and solid raw materials in a mass ratio of 80~90:10~20, wherein the solid raw materials include: 70-90 parts by weight of polymeric artificial bone matrix material; 5-20 parts by weight of hydroxyapatite; 0-10 parts by weight of metal phosphate, or 0-10 parts by weight of raw materials for preparing metal phosphate; 0-5 parts by weight of type I collagen / hyaluronic acid; The raw materials also include a pore-forming agent, the volume of which accounts for 80%-95% of the total volume of the pore-forming agent, the polymeric artificial bone matrix material, and the hydroxyapatite.

2. The polymeric artificial bone according to claim 1, characterized in that, The polymeric artificial bone matrix material is selected from one or more of polylactic acid-glycolic acid copolymer, polylactic acid, and polycaprolactone.

3. The polymeric artificial bone according to claim 1, characterized in that, The metal element in the metal phosphate is selected from at least one of Mg, Zn, Sr, Mn, Co, and Cu; The raw materials for preparing metal phosphates include soluble metal salts and soluble phosphates. The soluble metal salts are selected from chloride salts of Mg, Zn, Sr, Mn, Co, and Cu metals, and the soluble phosphates are sodium or potassium salts containing hydrogen phosphate or dihydrogen phosphate.

4. The polymeric artificial bone according to claim 1, characterized in that, In the aforementioned type I collagen / hyaluronic acid, the molecular weight of type I collagen is 100~300 kDa, and the molecular weight of hyaluronic acid is 800~1000 kDa.

5. The polymeric artificial bone according to claim 1, characterized in that, The pore-forming agent is selected from sodium chloride and sucrose.

6. The polymeric artificial bone according to claim 1, characterized in that, The solvent is selected from at least one of dichloromethane, trichloromethane, and N,N-dimethylformamide.

7. The polymeric artificial bone according to claim 1, characterized in that, The porosity of the polymer artificial bone is 80%-95%, and the pore size is 200-400μm.

8. A method for preparing polymeric artificial bone as described in any one of claims 1 to 7, characterized in that, Includes the following steps: A) Mix type I collagen / hyaluronic acid, hydroxyapatite, metal phosphate, polymeric artificial bone matrix material and solvent to obtain a mixture; B) Mix the mixture with the pore-forming agent to obtain a mixture; C) After molding the mixture, remove the pore-forming agent and freeze-dry it to obtain polymeric artificial bone; Alternatively, it may include the following steps: a) A mixture is prepared by mixing hydroxyapatite, metal phosphate, polymeric artificial bone matrix material, and solvent; b) Mix the mixture with the pore-forming agent to obtain a mixture; c) After molding the mixture, remove the pore-forming agent and freeze-dry it to obtain polymeric artificial bone; d) Dissolve type I collagen / hyaluronic acid in water once, immerse the artificial bone in the type I collagen / hyaluronic acid aqueous solution, freeze dry, and obtain polymeric artificial bone; Alternatively, it may include the following steps: 1) A mixture is prepared by mixing hydroxyapatite, polymeric artificial bone matrix material, and solvent; 2) The mixture is mixed with a pore-forming agent to obtain a mixture; 3) After the mixture is allowed to stand and solidify, the pore-forming agent is removed, and the mixture is freeze-dried to obtain polymeric artificial bone; 4) Dissolve type I collagen / hyaluronic acid, soluble metal salt, and soluble phosphate in water once to obtain a mixed solution; 5) Immerse the artificial bone in the mixed solution and freeze-dry to obtain biocompatible polymeric artificial bone.

9. The preparation method according to claim 8, characterized in that, Step A) includes: A1) Type I collagen / hyaluronic acid, hydroxyapatite, and metal phosphates are added to a mixed solvent and dispersed to obtain a mixed solution; A2) The polymeric artificial bone matrix material is mixed with the mixed solution to obtain a mixed solution.

10. The application of a polymeric artificial bone as described in any one of claims 1 to 7 in the preparation of tissue engineering scaffolds and drug sustained-release carriers.

Images