Bone repair stent for promoting diabetic bone defect repair

Abstract

The invention belongs to the technical field of medical biomaterial engineering application, and particularly relates to a bone repair scaffold for promoting diabetic bone defect repair. The stent main body is provided with a functional material, and the functional material comprises one or a combination of more than two of the following components: platelet-derived growth factors, copper ions and strontium ions; the weight part ratio of the platelet-derived growth factor to the copper ions to the strontium ions is (0.001-0.1): (0.005-1): (0.01-10). According to the bone repair scaffold provided by the invention, through the synergistic effect of the platelet-derived growth factor (PDGF-BB) and trace metal copper and strontium, the bone defect and osteoporotic bone defect of a diabetic patient are treated.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medical biomaterials engineering application, and particularly relates to a bone repair scaffold for promoting repair of diabetic bone defects. BACKGROUND

[0002] At present, diabetes has become one of the chronic diseases with the highest incidence, and the number of diabetic patients with bone defects caused by factors such as trauma and inflammation is also increasing. The prevalence of osteopenia and osteoporosis in diabetic patients is as high as 48-72%. Type 2 diabetes (T2DM) is a common chronic metabolic disease characterized by high blood sugar, accounting for more than 90% of diabetic patients. Due to high blood sugar, diabetic patients may affect the bone healing process and increase the risk of bone resorption. Under the pathological condition of diabetes, the microenvironment of bone defects changes, high blood sugar can cause inflammatory response and increase of reactive oxygen species (ROS) during bone regeneration, accompanied by mitochondrial dysfunction, increasing the incidence of nonunion and delayed union of fractures and hindering the process of bone reconstruction.

[0003] The bone healing process of normal bone defect patients can be divided into inflammation period (1-7d), cartilage callus formation period (2-3w), hard callus formation period (3-4 months) and remodeling period (several months to several years). In the first 1-3 days of injury, acute inflammation period, trauma signals activate immune cells, cause a chemical gradient of inflammatory factors, and recruit more immune cells to the wound, triggering an immune response aimed at clearing invading pathogenic microorganisms and tissue debris. Under abnormal metabolism of diabetes, acute inflammation period cannot rely on its own immune regulation function to terminate in time, resulting in a long-term inflammatory microenvironment in the local area, triggering pathological changes such as autologous tissue degradation and increased local reactive oxygen species. Under such circumstances, due to the abnormality of the immune system of diabetic patients, the susceptibility to local bacterial environment is enhanced, and problems such as delayed healing time and wound infection are prone to occur. Therefore, unlike healthy organisms, diabetic patients' bone tissue defect healing requires more precise and targeted treatment methods to achieve the purpose of controlling inflammation and promoting bone regeneration.

[0004] Clinically, diabetic patients with bone defects typically require comprehensive treatment measures: 1. Strict control of blood glucose levels through medication such as insulin, oral hypoglycemic agents, and dietary adjustments; 2. Prophylactic use of antibiotics or anti-inflammatory drugs to reduce the risk of infection at the bone defect site; 3. Implantation of biomaterials to promote healing of the bone defect. However, most common bone repair materials lack osteoinductive properties or use only one or two active substances, which cannot fully address the complex pathological conditions of bone defect repair in diabetic patients. For example, patent applications CN112451744B describe a 3D-printed enzyme-containing bioactive scaffold and its preparation method for a diabetic bone defect repair material; CN118846216A describes the construction of a drug delivery system for bone defects and a method for constructing the delivery body. Bone repair scaffolds need to mimic the natural bone structure, creating an environment similar to the extracellular matrix (EMC) to provide a favorable survival environment for cells and tissues. Currently, there are no commercially available bone defect repair materials that are effective and fully meet the specific pathological microenvironment of diabetic patients. Summary of the Invention

[0005] (a) Technical problems to be solved In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a bone repair scaffold that promotes the repair of bone defects in diabetic patients. Through the synergistic effect of platelet-derived growth factor (PDGF-BB) and trace amounts of copper and strontium, it can effectively promote the rate of vascularization and synergistically fight bacteria and inflammation, and can effectively treat bone defects and osteoporotic bone defects in diabetic patients.

[0006] (II) Technical Solution To achieve the above objectives, the main technical solutions adopted by this invention include: In a first aspect, the present invention provides a bone repair scaffold for promoting the repair of bone defects in diabetic patients, comprising a scaffold body having functional materials on the scaffold body, the functional materials comprising one or more of the following components: platelet-derived growth factor, copper ions and strontium ions; The platelet-derived growth factor, copper ions, and strontium ions are present in a weight ratio of 0.001–0.1:0.005–1:0.01–10.

[0007] The bone repair scaffold provided by the application treats bone defects and osteoporotic bone defects of diabetic patients through the synergistic effect of platelet-derived growth factor (PDGF-BB) and trace amounts of copper and strontium. PDGF-BB is a key participant in the early inflammatory stage of bone healing, which strongly promotes tissue regeneration and blood vessels, stimulates cell proliferation and migration, and induces H-type blood vessels and bone formation. PDGF-BB promotes the migration and differentiation of mesenchymal stem cells and bone marrow-derived endothelial progenitor cells by binding to its receptor PDGF receptor beta, and triggers the mitogen-activated kinase and phosphoinositide 3 kinase-Akt signal cascade. Copper ions have significant antibacterial, angiogenic and osteogenic effects; strontium ions activate signal pathways such as vascular endothelial growth factor through various mechanisms, regulate M2 polarization of macrophages, and their anti-inflammatory and antioxidant effects can reduce inflammatory response and reduce cell oxidative damage. Experiments show that increasing platelet-derived growth factor, copper ions and strontium ions in the bone repair scaffold has a significant effect on bone defects in the special pathological microenvironment of diabetic patients.

[0008] Optionally, the scaffold body comprises a porous polymer scaffold and a biological coating loaded on the porous polymer scaffold. The porous polymer scaffold is mainly made of strontium / copper-doped bioceramic microspheres and a polymer polymer.

[0009] Optionally, the biological coating comprises platelet-derived growth factor.

[0010] Optionally, the preparation method of the strontium / copper-doped bioceramic microspheres comprises the following steps: S1: configuring a bioceramic preparation solution with a calcium source and a phosphorus source, adding soluble copper salt and soluble strontium salt to the bioceramic preparation solution, preparing bioceramic co-precipitation powder by a solution co-precipitation method, and preparing the strontium / copper-doped bioceramic microspheres by adjusting the pH value and calcium / phosphorus ratio of the reaction process to prepare bioceramic co-precipitation powder of different phases.

[0011] The application particularly provides that the bioceramic microspheres are doped with strontium / copper, and the platelet-derived growth factor is arranged on the biological coating, so that the synergistic effect is better.

[0012] Optionally, the porosity of the porous polymer scaffold is 30% to 90%, and the pore size is 10 to 2000 μm.

[0013] Optionally, the thickness of the biological coating is 0.005 to 0.4 mm.

[0014] Optionally, the calcium source comprises one or more than two combinations of the following components: calcium nitrate, calcium chloride, calcium carbonate, and calcium hydroxide. The phosphorus source comprises one or more than two combinations of the following components: phosphoric acid, ammonium phosphate, pyrophosphate, disodium hydrogen phosphate, diammonium hydrogen phosphate. The strontium salt comprises one or more than two combinations of the following components: strontium nitrate, strontium chloride, strontium acetate, strontium sulfate; and the copper salt is one or more than two combinations of copper chloride, copper sulfate, copper nitrate, and copper acetate.

[0015] Optionally, the preparation method of the porous polymer scaffold comprises the following steps: S2: dissolving the polymer in an organic solvent, fully stirring until clear; adding the strontium / copper doped bioceramic microspheres into the polymer solution, fully stirring and mixing to obtain a 3D printing ink, and obtaining the porous polymer scaffold by 3D printing.

[0016] The porous polymer scaffold prepared by the method of the application realizes the precise design of the pore size distribution, connectivity and individual adaptation of the bone repair scaffold.

[0017] Optionally, the polymer in the application is one or more of polylactide, polyglycolide, polycaprolactone, poly(lactide-co-glycolide), poly(lactide-co-caprolactone), and poly(p-dioxanone).

[0018] Optionally, the preparation method of the scaffold body comprises the following steps: S3: immersing the porous polymer scaffold in a coating solution with PDGF-BB, repeatedly vacuuming to remove bubbles, and drying to obtain the scaffold body with a PDGF-BB coating.

[0019] Optionally, the coating solution further comprises one or more than two combinations of collagen, gelatin, chitosan, polyethylene oxide, polydopamine, cyclodextrin, fibrin glue, and tannic acid.

[0020] Optionally, the concentration of PDGF-BB in the coating solution is 0.002-5%, the immersion time is 0.5-24h, the vacuum degree is 5-100Pa, and the drying temperature is 15℃-150℃.

[0021] Optionally, the weight ratio of the bioceramics, the polymer and the platelet-derived growth factor is 50-90:10-50:0.001-0.1.

[0022] The bone repair scaffold for promoting the repair of diabetic bone defects of the application can treat the bone defects and osteoporotic bone defects of diabetic patients, has good tailorability, and is beneficial to the tailoring and matching of the shape and size of the defect site of the diabetic patient by the clinician.

[0023] (Three) beneficial effects The beneficial effects of the application are: The bone repair scaffold for promoting repair of diabetic bone defects of the application has a synergistic antibacterial and anti-inflammatory effect of platelet-derived growth factor (PDGF-BB) metal copper and strontium, and can precisely regulate the local microenvironment of the bone defect site. Specifically, the scaffold provided by the application can effectively deal with the problems of high inflammation, high infectivity and slow healing commonly found in bone defect areas. Through the in-situ regulation mechanism, it significantly optimizes these adverse conditions and accelerates the process of angiogenesis, which is crucial for improving the efficiency of tissue repair.

[0024] Among them, the bone healing ability of this particular group of diabetic patients is often weakened due to the influence of the disease, and the scaffold provided by the application can be particularly targeted at diabetic patients, not only promoting the rapid and effective healing of bone defects, but also having significant potential in treating osteoporotic bone defects.

[0025] Among them, the application further precisely controls the pore structure and suitable mechanical properties for optimal cell proliferation and differentiation in the design of the bone repair scaffold structure.

[0026] Among them, the scaffold of the application not only has matching mechanical properties of human bone, but also has a high proportion of bioceramics, which can effectively neutralize the acidic degradation products of PLGA and the acidic environment caused by inflammation in diabetic bone damage 。 BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is a model diagram of the bone repair scaffold of Example 6 of the application; Figure 2 is Figure 2 -A is a SEM image of the overall appearance of the bone repair scaffold with a coating thickness of 0 um; Figure 2 -B is a SEM image of the surface morphology of the bone repair scaffold with a coating thickness of 0 um; Figure 2 -C is a SEM image of the internal microsphere particle morphology of the bone repair scaffold with a coating thickness of 0 um; Figure 2 -D is a SEM image of the surface morphology of the bone repair scaffold with a coating thickness of 2 um; Figure 2 -E is a SEM image of the surface morphology of the bone repair scaffold with a coating thickness of 20 um; Figure 2 -F is a SEM image of the surface morphology of the bone repair scaffold with a coating thickness of 5 um; Figure 3 is the strontium and copper ion release curve of the bone repair scaffold of Example 6 in PBS solution; Figure 4 is a result graph of the antibacterial rate of each bone repair scaffold against Staphylococcus aureus under co-culture; Figure 5 pH variation curve of the bone repair scaffold of Example 6 of the present application in PBS solution (pH = 7.4); Figure 6 Degradation curve of the bone repair scaffold of Example 6 of the present application in PBS solution; Figure 7 Result graph of the proliferation of osteoblasts cultured by different bone repair scaffolds of the present application; Figure 8 Result graph of the relative expression amount of ALP of osteoblasts cultured by different bone repair scaffolds of the present application for 14 days; Figure 9 Result graph of the relative expression amount of IL-6 of macrophages cultured by different bone repair scaffolds of the present application for 5 days; Figure 10 Result graph of the relative expression amount of TNF-α of macrophages cultured by different bone repair scaffolds of the present application for 5 days. DETAILED DESCRIPTION

[0028] To make clear the possible application scenarios, technical principles, specific implementable schemes, and the purposes and effects achieved by the present application, the following will be described in detail in combination with the specific embodiments listed and the accompanying drawings. The embodiments described herein are only used to make the technical solutions of the present application clearer, and therefore only serve as examples, but cannot be used to limit the protection scope of the present application.

[0029] In this text, the term “embodiment” means that the specific features, structures or characteristics described in combination with the embodiments can be included in at least one embodiment of the present application. The term “embodiment” appearing at various positions in the specification does not necessarily refer to the same embodiment, and does not particularly limit the independence or association between the other embodiments. In principle, in the present application, as long as there is no technical contradiction or conflict, each technical feature mentioned in each embodiment can be combined in any way to form a corresponding implementable technical solution.

[0030] Unless otherwise defined, the meanings of the technical terms used herein are the same as those commonly understood by the person skilled in the art to which the present application belongs; the use of related terms herein is only for the purpose of describing specific embodiments, and is not intended to limit the present application.

[0031] In the description of the present application, the phrase “and / or” is a description of the logical relationship between the objects, which means that there can be three relationships, for example, A and / or B, which means that there are three cases: A exists, B exists, and A and B exist at the same time. In addition, the character “ / ” herein generally represents that the associated objects before and after are a “or” logical relationship.

[0032] In the present application, the terms such as "first" and "second" are merely used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is any actual number, primary or secondary, or order relationship between the entities or operations.

[0033] In the present application, the "includes", "contains", "has", or other similar expressions used in the statements are intended to cover non-exclusive inclusion, and these expressions do not exclude the presence of additional elements in the process, method or product comprising the elements, so that the process, method or product comprising a series of elements can not only include those defined elements, but also include other elements not explicitly listed, or also include elements inherent to such process, method or product.

[0034] As the same understanding as in the "Guidelines for Examination", in the present application, the expressions such as "greater than", "less than", "exceed" are understood as not including the number; the expressions such as "above", "below", "within" are understood as including the number. In addition, in the description of the embodiments of the present application, the meaning of "multiple" is more than two (including two), and similar expressions related to "multiple" are also understood in this way, for example, "multiple groups", "multiple times", etc., unless otherwise explicitly specified.

[0035] In the description of the embodiments of the present application, the spatial-related expressions used, such as "center", "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "vertical", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential", etc. The indicated orientation or position relationship is based on the orientation or position relationship shown in the specific embodiment or the drawing, and is only for the convenience of describing the specific embodiments of the present application or for the reader to understand, and does not indicate or imply that the indicated device or component must have a specific position, a specific orientation, or be constructed or operated in a specific orientation, and therefore cannot be understood as a limitation on the embodiments of the present application.

[0036] Unless otherwise explicitly specified or limited, in the description of the embodiments of the present application, the terms "mount", "connect", "connect", "fix", "set", etc. should be understood in a broad sense. For example, the "connection" can be a fixed connection, or a detachable connection, or an integral setting; it can be a mechanical connection, or an electrical connection, or a communication connection; it can be directly connected, or indirectly connected through an intermediate medium; it can be the internal communication of two elements or the interaction relationship between two elements. For those skilled in the art to which the present application belongs, the specific meaning of the above terms in the embodiments of the present application can be understood according to the specific circumstances. Embodiment 1

[0037] The embodiment provides a bone repair bracket for promoting repair of a diabetic bone defect, and the bracket body is provided with a functional material, and the functional material is platelet-derived growth factor, copper ions and strontium ions in a weight ratio of 0.001:1:5. Embodiment 2

[0038] The embodiment provides a bone repair bracket for promoting repair of a diabetic bone defect, and the bracket body is provided with a functional material, and the functional material is platelet-derived growth factor, copper ions and strontium ions in a weight ratio of 0.1:0.02:10. Embodiment 3

[0039] The embodiment provides a bone repair bracket for promoting repair of a diabetic bone defect, and the bracket body is provided with a functional material, and the functional material is platelet-derived growth factor, copper ions and strontium ions in a weight ratio of 0.08:0.005:0.01. Embodiment 4

[0040] The embodiment provides a more specific bone repair bracket for promoting repair of a diabetic bone defect relative to the embodiments 1-3, and the bracket body is composed of a porous high-molecular bracket and a biological coating loaded on the porous high-molecular bracket; the porous high-molecular bracket is made of strontium / copper-doped bioceramic microspheres and a high-molecular polymer. The biological coating is provided with platelet-derived growth factor.

[0041] The embodiment limits the platelet-derived growth factor to be arranged on the biological coating, limits the strontium and copper-doped bioceramic microspheres to be made into the porous high-molecular bracket, and the platelet-derived growth factor, the strontium ions and the cooper synergistically play a more significant effect relative to other methods. The other methods include but are not limited to: The strontium, the cooper and the platelet-derived growth factor are made into the porous high-molecular bracket; Or The strontium, the cooper and the platelet-derived growth factor are added on the biological coating; One of the strontium and the cooper and the platelet-derived growth factor are mixed and then added on the biological coating or made into the porous high-molecular bracket. Embodiment 5

[0042] The embodiment provides a preparation method of a bone repair bracket for promoting repair of a diabetic bone defect relative to the embodiment 4, and the preparation method comprises the following steps. Preparation of S1 Strontium / Copper Doped Bioceramic Microspheres: A bioceramic preparation solution was prepared using calcium and phosphorus sources. Soluble copper salt and soluble strontium salt were added to the bioceramic preparation solution. Bioceramic coprecipitate powder was prepared by solution coprecipitation. By adjusting the pH value and calcium / phosphorus ratio of the reaction process, bioceramic coprecipitate powders with different phases were prepared, and then the Strontium / Copper Doped Bioceramic Microspheres were obtained. Preparation of S2 porous polymer scaffold: The polymer is dissolved in an organic solvent and stirred thoroughly until clear; the strontium / copper doped bioceramic microspheres are added to the polymer solution and stirred thoroughly to obtain 3D printing ink; the porous polymer scaffold is obtained by 3D printing. Preparation of S3 scaffold body: The porous polymer scaffold is immersed in a coating solution with PDGF-BB, repeatedly vacuum defoamed, and dried to obtain the scaffold body with a PDGF-BB coating.

[0043] The porous polymer scaffold has a porosity of 30%–90% and a pore size of 10–2000 μm.

[0044] The thickness of the bio-coating is 0.005–0.4 mm.

[0045] In this embodiment, the bioceramic, polymer, and platelet-derived growth factor are in the following weight ratio: 90:10:0.1.

[0046] In some other specific embodiments, the bioceramic, polymer and platelet-derived growth factor are in a weight ratio of 80:40:0.001.

[0047] In some other specific embodiments, the bioceramics, polymers and platelet-derived growth factors are in a weight ratio of 50:50:0.05.

[0048] In some other specific embodiments, the concentration of the coating solution is 0.002-5%, the soaking time is 0.5-24h, the vacuum degree is 5-100Pa, and the drying temperature is 15℃-150℃. Example 6

[0049] This embodiment provides a more specific method for preparing a bone repair scaffold to promote the repair of bone defects in diabetic patients compared to Embodiment 5. The steps are as follows: Preparation of S1 Strontium / Copper Doped Bioceramic Microspheres: S11: 0.31 mol / L of calcium source solution was prepared using Ca(NO3)2 as the calcium source; part of the calcium source solution was used to dissolve strontium nitrate, so that the molar ratio of Sr / (Sr+Ca) was 10%, to obtain a strontium / calcium source mixed solution; part of the calcium source solution was used to dissolve copper nitrate, so that the molar ratio of Cu / (Cu+Ca) was 1%, to obtain a copper / calcium source mixed solution; the solutions were stirred at room temperature for 1 h using a magnetic stirrer until complete dissolution; S12: 0.2 mol / L of phosphorus source solution was prepared using (NH4)HPO4 as the phosphorus source; the phosphorus source solution was added at a constant rate to the strontium / calcium source mixed solution or the copper / calcium source mixed solution using a peristaltic pump (rate setting 20 mL / min); during the addition, the pH value was adjusted to about 8.6 using ammonia water; after the addition was completed, stirring was continued for 2 h, during which the pH value was maintained; then the solution was left to stand at room temperature for 24 h; after the addition, the molar ratio of (Sr+Ca) / P in the strontium / calcium source mixed solution and the molar ratio of (Cu+Ca) / P in the copper / calcium source mixed solution were both 1.55; S13: After the strontium / calcium source mixed solution and the copper / calcium source mixed solution were reacted in step S12, they were left to stand for 1-2 h, so that the reactants precipitated; then the supernatant was poured off; the precipitates were collected and washed by suction filtration in a Buchner funnel; the filter cake was added to about 20 times the volume of pure water; the mixture was stirred and dispersed, then suction filtered and washed again; this process was repeated three times to remove residual ions; strontium / calcium source white precipitate paste and copper / calcium source white precipitate paste were obtained, respectively; S14: The strontium / calcium source white precipitate paste and the copper / calcium source white precipitate paste after centrifugal washing were placed in a constant temperature drying oven and dried at 50°C for 12 h to obtain strontium / calcium source white solids and copper / calcium source white solids; the strontium / calcium source white solids and the copper / calcium source white solids were crushed and calcined in a muffle furnace, with a temperature rise rate of 6°C / min; the temperature was held at 900°C for 2.5 h; Sr-BCP powder and Cu-BCP powder with a HA:β-TCP phase ratio of 7:3 were obtained, respectively.

[0050] S15: Spray drying: Preparation of Sr-BCP microspheres: 1% polyvinyl alcohol, Sr-BCP powder and water were mixed to obtain a slurry with a solid content of 10%. The Sr-BCP slurry was spray dried using a spray dryer, with a spray drying temperature of 200°C, a feed rate of 2000 mL / h, and a nozzle diameter of 0.5 mm; then sintering was performed in a muffle furnace at a sintering temperature of 500°C for 1 h; Sr-BCP microspheres with a porous structure were obtained; The preparation method of the Cu-BCP microspheres is the same as that of the Sr-BCP microspheres described above, except that the Sr-BCP powder is replaced by Cu-BCP, to obtain Cu-BCP microspheres with a porous structure.

[0051] The Sr-BCP microspheres and the Cu-BCP microspheres are classified by sieving the crude product after calcination with a screen, to obtain hydroxyapatite microspheres with a particle size distribution of 15-30 μm.

[0052] S2: 2 g of PLGA is dissolved in 20 mL of dichloromethane, and stirred for 3 h to obtain a clear polymer solution. Then, 5 g of Sr-BCP microspheres and 3 g of Cu-BCP microspheres are added, and after closed ultrasonic stirring for 1 h, mechanical stirring is performed at room temperature, while maintaining an open environment to allow the solvent to evaporate to achieve a solid content of 50%, to obtain a printing ink.

[0053] The printing is performed at room temperature 23 ℃, the printing substrate is a polytetrafluoroethylene coated substrate, the printing pressure is 0.6 MPa, the printing speed is 50 mm / s, the printing wire diameter is 410 um, the printing angle is 0-90°, 10*10*20 mm, and the printing pitch is 800 um, to obtain a bone repair scaffold blank; then the scaffold blank is placed in a 30 ℃ air drying oven for drying for 48 h, to obtain a strontium / copper doped porous polymer scaffold.

[0054] S3: Preparation of a scaffold coating: a gelatin coating solution with a concentration of 0.5% and containing 0.01% PDGF-BB is prepared, and the bone repair scaffold body is immersed in the coating solution, and vacuum degassing is repeatedly performed at a vacuum degree of 20 Pa, and then the scaffold is taken out and air dried at 30 ℃, to obtain a bone repair scaffold for promoting the repair of diabetic bone defects, with a bio-coating thickness of 0.02 mm. Example 7

[0055] This example provides a method for preparing a bone repair scaffold for more specifically promoting the repair of diabetic bone defects, relative to Example 5, and the steps are as follows: S1: Preparation of strontium / copper doped bioceramic microspheres: S11: A calcium nitrate tetrahydrate solution is prepared at a concentration of 113 g / L, as a calcium source solution; Part of the calcium source solution is used to dissolve strontium nitrate, so that the molar ratio of Sr / (Sr+Ca) is 7%, to obtain a strontium / calcium source mixed solution; Part of the calcium source solution is used to dissolve copper nitrate, so that the molar ratio of Cu / (Cu+Ca) is 2%, to obtain a copper / calcium source mixed solution. The above solutions are all stirred at room temperature with a magnetic stirrer for 1 h to completely dissolve them; S12: Synthesis: The sodium phosphate dibasic dodecahydrate solution was prepared as a phosphorus source solution at a concentration of 171.38 g / L. The phosphorus source solution was added to the strontium / calcium source mixed solution or the copper / calcium source mixed solution at a constant rate by using a peristaltic pump (rate setting 20 mL / min). During the addition, sodium hydroxide was used to adjust the pH value to maintain it at about 7.4. After the addition was completed, stirring was continued for 2 h, and then the solution was left to stand at room temperature for 48 h. During this period, the pH value was maintained. After the addition, the molar ratio of (Sr+Ca) / P in the strontium / calcium source mixed solution and the molar ratio of (Cu+Ca) / P in the copper / calcium source mixed solution were both 1.67. S13 Washing: After the strontium / calcium source mixed solution and the copper / calcium source mixed solution were left to stand for 1-2 h after the reaction in step S12, the reactants were precipitated, and then the supernatant was discarded. The precipitates were collected and washed by suction filtration in a Buchner funnel. The filter cake was collected and washed by stirring and dispersing in about 20 times the volume of pure water. The washing was repeated three times to remove residual ions. The strontium / calcium source white precipitate paste and the copper / calcium source white precipitate paste were obtained. S14 Drying: The strontium / calcium source white precipitate paste and the copper / calcium source white precipitate paste were collected and spread on a freeze-drying tray, and then placed in a freeze-drying machine for freeze-drying. The thickness of the material was as low as possible to facilitate water removal. The freeze-drying parameters were as follows: -35 °C, 1 h for cooling, 4 h for maintaining; -10 °C, 30 min for heating, 15 h for maintaining; 25 °C, 30 min for heating, 8 h for maintaining. After freeze-drying, the material was collected and ground and sieved to obtain Sr-HA powder and Cu-HA powder.

[0056] S15 Spray drying: Preparation of Sr-HA microspheres: 1% polyvinyl alcohol, Sr-HA powder, and water were mixed to obtain a slurry with a solid content of 15%. The Sr-HA slurry was spray dried using a spray dryer at a temperature of 200 °C, a feeding speed of 20 mL / min, and a nozzle diameter of 0.3 mm. Then, the product was sintered in a muffle furnace at a sintering temperature of 700 °C for 1 h to obtain Sr-HA microspheres with a porous structure. The preparation method of Cu-HA microspheres was the same as that of Sr-HA microspheres described above, except that the Sr-HA powder was replaced by Cu-HA powder to obtain Cu-HA microspheres with a porous structure. The calcined hydroxyapatite microspheres were classified and sieved using a screen to obtain hydroxyapatite microspheres with a particle size distribution of 10-20 μm.

[0057] S2: 1.765 g PLGA was dissolved in 10 mL dichloromethane, stirred for 3 h to obtain a clear polymer solution. Then 7 g Sr-HA microspheres and 3 g Cu-HA microspheres were added, and after 1.5 h of closed ultrasonic stirring, mechanical stirring was carried out at room temperature, and the environment was kept open to allow the solvent to evaporate to a solid content of 50% to obtain a printing ink.

[0058] Printing was carried out at room temperature 23°C, the printing substrate was a polytetrafluoroethylene coated substrate, the printing pressure was 0.4 MPa, the printing speed was 20 mm / s, the printing wire diameter was 240 um, the printing angle was 0-120°, 10*10*20 mm, the printing pitch was 600 um, and a bone repair scaffold blank was prepared; then the scaffold blank was placed in a 30°C air drying oven and dried for 36 h to obtain a strontium / copper doped porous polymer scaffold; S3: A gelatin coating solution with a concentration of 0.3% was prepared, and 0.005% PDGF-BB was added to the solution. The bone repair scaffold was immersed in the coating solution, and vacuum was repeatedly applied to remove bubbles. The vacuum degree was 20 Pa, and then the scaffold was taken out and dried at 50°C to obtain a bone repair scaffold for promoting the repair of diabetic bone defects. Example 8

[0059] This example provides a method for preparing a bone repair scaffold that more specifically promotes the repair of diabetic bone defects, relative to Example 5, and the steps are as follows: S1: Preparation of strontium / copper doped bioceramic microspheres: S11: A calcium nitrate tetrahydrate solution with a concentration of 113 g / L was prepared as a calcium source solution. Strontium nitrate and copper nitrate were dissolved in the calcium source solution to obtain a strontium / copper / calcium source mixed solution, with a Sr / (Sr+Cu+Ca) molar ratio of 10% and a Cu / (Sr+Cu+Ca) molar ratio of 2%. The above solutions were stirred at room temperature for 1 h using a magnetic stirrer to ensure complete dissolution. S12: A disodium hydrogen phosphate dodecahydrate solution with a concentration of 171.38 g / L was prepared as a phosphorus source solution. The phosphorus source solution was added to the strontium / copper / calcium source mixed solution at a constant rate of 25 mL / min using a peristaltic pump. During the addition process, the pH value was adjusted to be around 10 using sodium hydroxide. After the addition was completed, the solution was stirred for another 2 h, and then it was left to stand at room temperature for 24 h. During this period, the pH value was maintained. After the addition, the (Sr+Cu+Ca) / P molar ratio in the strontium / copper / calcium source solution was 1.67. S13 washing: the reaction solution obtained after step S12 reaction is allowed to stand for 1-2 h to make the reactants precipitate, and then the supernatant is poured off, and the precipitate is subjected to suction filtration and washing in a Buchner funnel, the filter cake is collected, about 20 times the volume of pure water is added to the filter cake, and the mixture is stirred and dispersed for washing, and then suction filtration and washing are performed again, and the process is repeated three times to remove residual ions, to obtain a white precipitate paste.

[0060] S14 drying: the white precipitate paste is collected, spread on a freeze-drying tray, and placed in a freeze-drying machine for freeze-drying; attention is paid to making the material thickness as low as possible to facilitate water removal. Freeze-drying parameters: -25°C cooling time 1 h, holding time 5 h; -10°C warming time 15 min, holding time 10 h; 25°C warming time 35 min, holding time 16 h. The freeze-dried material is collected and crushed and sieved to obtain SrCu-HA powder; S15 spray drying: 1% polyvinyl alcohol, SrCu-HA powder and water are mixed to obtain a slurry with a solid content of 30%. The SrCu-HA slurry is spray dried using a spray dryer, the spray drying temperature is 200°C, the feeding speed is 20 mL / min, and the nozzle diameter is 0.7 mm. Then it is placed in a muffle furnace for sintering, the sintering temperature is 600°C, and the sintering time is 1 h, to obtain SrCu-HA microspheres with a porous structure. The crude SrCu-HA microspheres after calcination are sieved using a screen to obtain hydroxyapatite microspheres with a particle size distribution of 15-35 μm; S2 2.4 g of PLGA is dissolved in 30 mL of dichloromethane, stirred for 3 h to obtain a clear polymer solution. Then 6.8 g of SrCu-HA microspheres are added, and after ultrasonic stirring for 2 h, mechanical stirring is carried out at room temperature while maintaining an open environment to allow the solvent to evaporate to a solid content of 40% to obtain a printing ink. Print at room temperature 25°C, print the bottom plate PTFE coated substrate, print pressure 0.32 MPa, print speed 30 mm / s, print wire diameter 410 um, print angle 0-90°, 10*10*20 mm, print spacing 410 um, to prepare a bone repair scaffold blank; then the scaffold blank is placed in a 37°C air drying oven and dried for 48 h to obtain a strontium / copper doped porous polymer scaffold.

[0061] S3 scaffold coating preparation: a gelatin coating solution with a mass concentration of 0.01% PDGF-BB is prepared, wherein the gelatin concentration is 0.5%, the bone repair scaffold body is immersed in the coating solution, vacuum is repeatedly applied to remove bubbles, the vacuum degree is 20 Pa, then it is taken out and air dried at 30°C to obtain a bone repair scaffold for promoting the repair of diabetic bone defects.

[0062] Comparative Examples 1-10 provide a method for preparing a bone repair scaffold for promoting repair of bone defects in diabetes, the steps of which are the same as Example 6, and the differences of each comparative example are shown in Table 1: Table 1

[0063] Experiments show that the bone repair scaffolds prepared in Examples 1-10 can promote the repair of bone defects in diabetic patients, in particular, strontium, copper and platelet-derived growth factor have a synergistic effect on the speed and effect of promoting bone defect repair, in addition, the thickness of the coating in the scaffold structure of the application and the porous pore ratio have a significant influence on the above-mentioned synergistic effect, only within the range defined in the application can have the optimal effect.

[0064] In the present application: (1) The additive manufacturing multilayer circulating bone repair scaffold has good toughness and tailorability, and in clinical practice, it can be implanted by simple cutting and pressing after precise matching of the bone defect site of the patient.

[0065] (2) The multilayer 10-80° angle designed bone repair material structure prepared by the additive manufacturing process provides the best pore size space for cell crawling proliferation and bone tissue ingrowth, promotes cell migration and nutrient distribution, promotes osteogenesis and angiogenesis, and completes tissue regeneration and repair.

[0066] (3) By controlling the use of uniform particle size microspheres, the product preparation process is effectively optimized, the printing process is smoother, continuous and stable printing is achieved, and the industrialization difficulty is low.

[0067] (4) By using microspheres with uniform particle size, a better polymer network structure is obtained, thereby improving the overall microscopic consistency of the product and the mechanical properties of the bone repair material. Without crosslinking and grafting of raw materials, the process is simple and has high biological safety. The compressive strength can reach 6-15 MPa, and the Young's modulus can reach 30-60 MPa, accurately matching the mechanical requirements of cancellous bone repair.

[0068] (5) The scaffold of the present application has a high bioceramic ratio while matching the mechanical properties of human bone, and can effectively neutralize the acidic degradation products of PLGA in the scaffold and the acidic environment caused by inflammation in diabetic bone damage during the degradation and repair process.

[0069] In the present application, the gelatin coating provides hydrophilicity, improves blood infiltration, is conducive to cell proliferation adsorption and transfer, and the PDGF in the coating plays a role in the inflammation stage. At the same time, the microspheres in the stent can play a slow-release role of strontium and copper after being in contact with body fluids, thereby achieving antibacterial, anti-inflammatory, pro-vascular, pro-bone growth, and pro-bone cell repair from various aspects. The slow-release effect of the microspheres can reduce the cytotoxicity and other dose hazards caused by the one-time release of trace metals.

[0070] 1. Microsphere, stent sample morphology experiment Experimental method: Scanning electron microscope (SEM) was used to observe the differences in the microstructure of the stent with different coating thicknesses. The bone repair stent was cut into a cross section of the bone repair stent coating with a scalpel, and then the surface was pasted onto a sample stage. Gold was sprayed to observe the coating condition and thickness of the coating covering the stent, and the microstructure image of the sample was photographed and the coating thickness was calculated.

[0071] The tested sample was the bone repair stent for promoting the repair of diabetic bone defects in Example 6, and by adjusting the number of times the bone repair stent body was soaked in the coating solution in step S3, bone repair stents with different thicknesses were obtained, which were 0 um, 2 um, 5 um, and 20 um, respectively.

[0072] Figure 2 -A is the SEM image of the overall appearance of the bone repair stent with a coating thickness of 0 um. It can be seen from the image that the surface of the uncoated stent is relatively rough, and the texture of the concave and convex surface formed after drying of the composite stent can be observed on the surface.

[0073] Figure 2 -B is the SEM image of the surface morphology of the bone repair stent with a coating thickness of 0 um; Figure 2 -C is the SEM image of the internal microsphere particle morphology of the bone repair stent with a coating thickness of 0 um; Figure 2 -D is the SEM image of the surface morphology of the bone repair stent with a coating thickness of 2 um; Figure 2 -E is the SEM image of the surface morphology of the bone repair stent with a coating thickness of 5 um; Figure 2 -F is the SEM image of the surface morphology of the bone repair stent with a coating thickness of 20 um; Figure 2 In B and 2-C, it can be seen that the microspheres are uniformly dispersed on the surface and inside the stent, and many micropores can be observed in the microspheres and the high molecular matrix stent. Figure 2 As shown in D, 2-E, and 2-F, the surface morphology before and after coating modification is compared, and the surface is changed from rough to smooth after coating modification. The surface coating is uniform and dense, and there is no hole and crack structure.

[0074] 2. Bone repair scaffold activity map: antibacterial effect experiment.

[0075] Experimental method: cut the bone repair scaffold sample into 5*5*2mm, sterilize it by ultraviolet rays, then place it in a sterile centrifuge tube, take 1mL of culture medium, 100uL of staphylococcus aureus suspension (1x10 5 cfu / mL) and add it, place it in a constant temperature shaker, incubate at 37℃ for 24 hours. Take the co-cultured bacterial solution, perform gradient dilution, select an appropriate dilution factor for plate coating. Uniformly coat 100uL of the diluted bacterial solution on the agar medium, set 3 parallel samples for each concentration gradient. Place the plate in a 37℃ constant temperature incubator and incubate for 24h, then count the colonies after colony formation. Control group: do not add samples, only add bacterial suspension, plate count after 24 hours of 37℃ constant temperature incubation. Calculate the antibacterial rate by plate count method, the formula is as follows: antibacterial rate = [(control group bacterial colony number-experimental group bacterial colony number) / control group bacterial colony number]x100%.

[0076] Take staphylococcus aureus as the determination index, and perform antibacterial research on the bone repair scaffold obtained in Example 6, and the antibacterial performance results are as shown in Figure 4 The antibacterial rate of Example 6 is 90±3.2%. And the scaffolds of Comparative Example 2, Comparative Example 3, Comparative Example 7, Comparative Example 9 all have obvious antibacterial effect, which are 72.2±1.32%, 89.8±1.8%, 74.9±2.89%, 81.2±0.59% respectively. While Comparative Example 1, Comparative Example 4, Comparative Example 5, Comparative Example 6, Comparative Example 8, Comparative Example 10, Comparative Example 11 also show a small amount of antibacterial property, which may be because Sr also has a small amount of antibacterial activity, and the scaffold has a certain adsorption to the bacteria in the bacterial solution. The results show that the bone repair scaffold of the present application has obvious antibacterial effect.

[0077] From the comparison of Comparative Example 7 and Comparative Example 9, it can be seen that: the thickness of the biological coating is too thick (0.4mm of Comparative Example 7), which significantly reduces the antibacterial performance.

[0078] From the comparison of Comparative Example 2 and Comparative Example 9, it can be seen that: the addition of PDGF-BB in Comparative Example 2 significantly reduces the antibacterial performance; but from the comparison of Example 6 and Comparative Example 3, it can be seen that: the addition of PDGF-BB has no effect on the antibacterial performance; thus it is analyzed that Example 6 and Comparative Example 3 have strontium nitrate, which reacts with PDGF-BB, eliminating the negative effect of PDGF-BB on the antibacterial performance.

[0079] From the comparison of Example 6, Comparative Example 1, Comparative Example 2 and Comparative Example 4, it can be seen that: the addition of strontium nitrate itself does not contribute to the antibacterial performance, but has a positive synergistic effect on the antibacterial performance of strontium nitrate.

[0080] From each pair of proportions can be seen: the addition of PDGF-BB in the gelatin coating solution has no effect on the antibacterial performance; From the antibacterial effect comparison of Example 1 and Comparative Example 2, the addition amount of strontium nitrate has a significant effect on the antibacterial performance.

[0081] 3. Porosity and mechanical data Experimental method: The porosity of the bone repair scaffold was measured by the Archimedes drainage method. The dry weight M0 of the bone repair scaffold sample was weighed, then the sample was immersed in pure water and placed in a vacuum drying oven for 30 min to exhaust the bubbles in the scaffold. Then the suspended weight M1 of the sample after water saturation in water and the mass M2 of the sample after water saturation taken out in air were weighed, and the porosity was calculated according to the formula. The formula is as follows: Porosity = [(M2-M0) / (M2-M1)]x100% The compressive strength of the bone repair scaffold was tested by using an electronic universal testing machine. The standard cuboid sample with the size of 10*10*15 mm was prepared, and the test speed was 1 mm / min. From the beginning of the test to the rupture of the sample, the load displacement curve was drawn, and the maximum load and maximum load displacement in the load displacement curve of each sample were recorded. The maximum load was used to calculate the compressive strength. At the same time, the Young's modulus was calculated according to the first 5% strain range. Table 2 lists the porosity, compressive strength and Young's modulus of the bone repair scaffold. It can be found that the scaffold has good compressive strength while maintaining high porosity. The porosities of Example 6, Example 7 and Example 8 are 81.40±0.85%, 87.1±1.35%, 67.3±1.51% respectively, which are slightly larger than the filling rate set by the printing parameters, because the scaffold shrinks after drying and a large number of micropores are formed after drying. The compressive strengths of Example 6, Example 7 and Example 8 are 9.52±0.72 MPa, 6.77±0.45 MPa, 14.73±1.39 MPa respectively, and the Young's moduli are 31.92±3.47 MPa, 25.39±2.98 MPa, 34.16±5.47 MPa respectively. From the mechanical data of the scaffold, it can be seen that the compressive strength and Young's modulus of the scaffold are close to the cancellous bone of human bone and higher than other degradable bone repair products, so it can provide a similar mechanical environment for the bone repair process.

[0082] Table 2 Porosity and mechanical property data table of the scaffold

[0083] 4. In vitro degradation data graph-ph change, degradation rate Experimental method: Cumulative PH change test: Prepare 10*10*10mm standard cuboid samples, 3 parallels for each time point, use 1 container for each parallel sample, the test sample should be completely immersed in the test solution PBS (pH=7.4), the ratio of the volume (mL) of the test solution to the mass (g) of the test sample is 100:1, and the volume of the test solution is not less than 10mL. The constant temperature shaker is kept at 37℃, and the rotation speed is 60rpm. Measure the solution PH value according to the time point and record the data of each time point, and draw the curve.

[0084] Degradation test: Prepare 10*10*10mm standard cuboid samples, 3 parallels for each time point, use 1 container for each parallel sample, the test sample should be completely immersed in the test solution PBS (pH=7.4), the ratio of the volume (mL) of the test solution to the mass (g) of the test sample is 100:1, and the volume of the test solution is not less than 10mL, and the simulated solution is replaced every 72h. The constant temperature shaker is kept at 37±1℃, and the rotation speed is 60rpm. According to the time point, take out the sample, dry the degraded sample at 50℃ under vacuum to constant weight, and calculate the mass loss of the sample before and after degradation.

[0085] The pH change and degradation performance results of the bone repair scaffold are shown in Figure 5 , Figure 6 The pH change curve of the scaffold of Example 6 in PBS solution (pH=7.4) is shown in Figure 5 From the curve, the pH of the scaffold remains stable overall, with a small fluctuation during the degradation of the scaffold, but the high proportion of bioceramics in the scaffold can effectively neutralize the acidic degradation products of PLGA to maintain around 7.4. As shown in Figure 6 The bone repair scaffold has a faster degradation rate in the first 7 days, mainly because the uncrosslinked gelatin coating degrades rapidly, and then enters the slow degradation interval, with a cumulative degradation of 29.33%. The results show that the high bioceramics of the bone repair scaffold of the application can effectively neutralize the acidic reaction in the environment, which is conducive to improving the acidic degradation products of PLGA and the acidic environment caused by inflammation in diabetic bone damage; in addition, the degradation time matches the repair time of human bone for 3-6 months.

[0086] 5. Osteoblast proliferation results and related gene expression chart--rBMSCs cells Experimental method: Osteoblast proliferation: 10*2 mm bone repair scaffold round samples were prepared and sterilized by ultraviolet rays. The prepared sterile scaffolds of each group were placed at the bottom of a 48-well plate and soaked with complete culture medium for 12 h. rBMSCs were digested to prepare a cell suspension, and the cell suspension was slowly dropped onto the surface of the scaffold at a density of 2*104 cells / well for co-culture. Fresh culture medium was replaced every other day. When cultured to the specified day, 10% CCK-8 reagent-containing medium was added to each well, and incubated in the dark for 2 h, followed by aspirating the reaction solution to a 96-well plate for measuring the absorbance at 450 nm by an enzyme labeler.

[0087] ALP activity test: 10*2 mm bone repair scaffold round samples were prepared and sterilized by ultraviolet rays. The prepared sterile scaffolds of each group were placed at the bottom of a 48-well plate and soaked with complete culture medium for 12 h. rBMSCs were digested to prepare a cell suspension, and the cell suspension was slowly dropped onto the surface of the scaffold at a density of 2*104 cells / well for co-culture. After 24 h of culture, osteogenic induction liquid was replaced, and replaced every other day. After 14 days of co-culture, the culture medium was discarded, washed once with PBS, and a sufficient amount of lysis solution was added to immerse the scaffold. After complete lysis, the lysis solution was obtained, and the ALP expression amount was determined by an ALP kit. 4

[0088] To evaluate the osteoblast proliferation results and related gene expression of the bone repair scaffolds (Example 6, and bone repair scaffolds prepared in Comparative Examples 1-10) of each group, the scaffolds of each group were co-cultured with rBMSCs for evaluation. As shown in Table 3, the CCK-8 experiment results showed that there was no significant difference in the proliferation of cells co-cultured with the bone repair scaffolds of each group on the first day, and the number of rBMSCs significantly increased as the culture time of the cells was prolonged, further indicating that the scaffolds prepared in the present study had good cell compatibility. On the third day and the seventh day of co-culture, the Example 6 group gradually showed a more obvious proliferation rate, which indicated that the bone repair scaffolds of the present application effectively promoted the proliferation of rBMSCs. Figure 7

[0089] Table 3 Proliferation of osteoblasts cultured with different scaffolds (OD value)

[0090] ​​Example 6 (containing strontium doping, copper doping, and PDGF-BB coating) exhibited the highest OD values ​​at all time points (1d, 3d, 7d) and maintained a steady increase over time, indicating that it significantly promoted cell proliferation and long-term viability. The OD values ​​of all comparative examples (1-8) were lower than those of Example 6, especially Comparative Example 5 (without strontium, copper, or PDGF-BB), which had the lowest value (0.805) at 7d, indicating that cell activity was severely impaired in the absence of key components. The OD values ​​of all groups increased from 1d to 7d, but Example 6 showed the largest increase, demonstrating that its effect was not only significant but also sustainable.

[0091] Comparative Example 1 (Copper-free): The OD value was significantly lower than that of Example 6, but higher than that of Comparative Example 4 (Strontium-free and Copper-free). For example, at 7 days, the OD value of Comparative Example 1 was approximately 1.25, while that of Comparative Example 4 was only approximately 0.75. This indicates that copper doping contributes independently to cell proliferation, but the presence of strontium can partially compensate for the deficiency. Comparative Example 2 (Strontium-free): The OD value was slightly higher than that of Comparative Example 1 (Copper-free), but much lower than that of Example 6. For example, at 3 days, the OD value of Comparative Example 2 was approximately 0.8, while that of Example 6 was approximately 1.8. This indicates that strontium doping is more critical for enhancing cell viability, but copper can enhance its effect. Comparative Example 4 (Strontium-free and Copper-free): One of the lowest OD values ​​(close to Comparative Example 5), indicating that the co-deficiency of strontium and copper leads to a significant decrease in cell activity. Strontium (Sr) and copper (Cu) ions may enhance cell metabolism in a diabetic environment by promoting angiogenesis and anti-inflammatory effects.

[0092] Comparative Example 7 (no strontium and no PDGF-BB, but with a thick coating of 0.4 mm and copper): The OD value was lower than that of Comparative Example 2 (no strontium but with standard PDGF-BB), indicating that simply increasing the coating thickness cannot compensate for the lack of key components, and may affect the porosity of the scaffold due to excessive thickness, thus hindering cell attachment.

[0093] The test results of ALP are as follows Figure 8 As shown in Table 4: Table 4. Relative ALP expression levels in osteoblasts cultured on different scaffolds for 14 days.

[0094] ALP is an early osteogenic marker of cell maturation and calcification. For example... Figure 8 As shown in Table 4, after 14 days of co-culture, the ALP activity of rBMSCs co-cultured with the Example 6 scaffold was significantly higher than that of other scaffolds. Furthermore, compared to Comparative Example 5, which was undoped with copper ions, strontium ions, and PDGF-BB coating, the ALP activity of other scaffold groups was increased to varying degrees. These results indicate that, under the synergistic effect of platelet-derived growth factor (PDGF-BB) and trace amounts of copper and strontium, the porous bone repair scaffold of Example 6 can provide a better environment for the proliferation and differentiation of rBMSCs.

[0095] Example 6 has the highest relative ALP expression (about 5), significantly better than all the comparative examples (1-8 are all in the range of 1-4), indicating its optimal osteogenic differentiation ability.

[0096] Comparative examples 1-8 all have low ALP values, especially comparative example 4 (no strontium no copper) and comparative example 5 (no all components) have the lowest (about 1), indicating that the osteogenic ability is severely impaired.

[0097] Strontium and copper synergistically affect bone differentiation: Comparative example 1 (no copper): ALP value is about 3, much lower than example 6 (about 5). Copper ions (Cu) can protect osteoblasts in a diabetic environment through antibacterial and pro-vascular effects.

[0098] Comparative example 2 (no strontium): ALP value is about 4, higher than comparative example 1 (no copper), but significantly lower than example 6. Strontium ions (Sr) are known osteogenic promoters (mimic calcium ion signals), and their absence directly affects ALP expression.

[0099] Comparative example 4 (no strontium no copper): ALP value (about 2.25), confirming that the absence of strontium and copper almost completely inhibits osteogenic differentiation.

[0100] In comparative example 5, there is no strontium, no copper, and no PDGF-BB, and the ALP value (about 1.0) compared to comparative example 4 shows that PDGF-BB significantly affects osteogenic differentiation.

[0101] The high ALP of example 6 (about 5) demonstrates the synergy of strontium, copper, and PDGF-BB: strontium / copper microspheres provide ion sustained release, enhancing cell adhesion; PDGF-BB coating directly stimulates differentiation.

[0102] Comparative example 7 (no strontium no PDGF-BB, but with copper and thick coating) has an ALP value of only about 2, indicating that a single component (such as copper) cannot independently drive osteogenesis.

[0103] 6. The scaffold reduces the expression of related inflammatory genes of macrophages.

[0104] Experimental method: Prepare 10*2 mm bone repair scaffold round samples, sterilize them with ultraviolet light. Place the prepared sterile scaffolds of each group at the bottom of the 48-well plate, soak them in fresh culture medium for 12 h. Seed RAW 264.7 in the well plate at a density of 2*104 cells / well, cultivate in a cell incubator for 5d, collect the supernatant, and detect the expression level of TNF-α, IL-6 in the supernatant by ELISA kit.

[0105] Bone fracture healing is carried out under the control of the immune system. During bone healing, immune cells infiltrate the hematoma and release cytokines, triggering inflammation. In the early stage after injury, M1 macrophages are the main population at the injury site, which secrete pro-inflammatory cytokines to cause local inflammation, such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), etc., which help to remove damaged tissues and recruit stem cells. However, long-term inflammation is harmful and is not conducive to tissue regeneration. Therefore, timely transition to M2 macrophages is a necessary condition for effective tissue regeneration.

[0106] From Figure 9 , Table 5 and Figure 10 , Table 6, compared with Comparative Example 5 without doping copper ions, strontium ions and PDGF-BB coating, the other scaffolds inhibit the secretion of IL-6 and TNF-a by RAW264.7 cells, and the inhibition degree of Example 6 is more significant. The final result shows that the scaffold doped with copper ions, strontium ions and PDGF-BB coating can significantly and effectively reduce the secretion amount of inflammatory factors, which is conducive to relieving the inflammatory reaction and creating a good immune microenvironment for diabetic bone defect tissue regeneration. In addition, copper ions, strontium ions and PDGF-BB have a synergistic effect on reducing the secretion amount of inflammatory factors.

[0107] Table 5 Relative expression amount of IL-6 of macrophages cultured for 5 days by different scaffolds

[0108] Table 6 Relative expression amount of TNF-α of macrophages cultured for 5 days by different scaffolds

[0109] In summary, the platelet-derived growth factor (PDGF-BB), metal copper and strontium in the application overcome side effects and have a synergistic effect on improving antibacterial and anti-inflammatory effects, and can precisely regulate the local microenvironment of the bone defect site. The coating thickness must be limited within a specific range, which further promotes the synergistic effect between platelet-derived growth factor (PDGF-BB), metal copper and strontium. That is, on the basis of the antibacterial and anti-inflammatory synergistic effect of platelet-derived growth factor (PDGF-BB), metal copper and strontium, the combination of the technical means of limiting the coating thickness can further significantly improve the antibacterial and anti-inflammatory synergistic effect.

[0110] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions recorded in the above embodiments can be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A bone repair scaffold for promoting the repair of bone defects in diabetic patients, comprising a scaffold body, characterized in that: The stent body has functional materials, which include one or more of the following components: platelet-derived growth factor, copper ions, and strontium ions; The platelet-derived growth factor, copper ions, and strontium ions are present in a weight ratio of 0.001–0.1:0.005–1:0.01–10.

2. The bone repair scaffold for promoting the repair of diabetic bone defects as described in claim 1, characterized in that, The scaffold body includes a porous polymer scaffold and a bio-coating loaded on the porous polymer scaffold; The porous polymer scaffold is mainly made of strontium / copper doped bioceramic microspheres and polymers.

3. The bone repair scaffold for promoting the repair of diabetic bone defects as described in claim 1, characterized in that: The bio-coating includes platelet-derived growth factor.

4. The bone repair scaffold for promoting the repair of diabetic bone defects as described in claim 2, characterized in that, The preparation method of the strontium / copper doped bioceramic microspheres includes the following steps: S1 prepares a bioceramic preparation solution using calcium and phosphorus sources, adds soluble copper and soluble strontium salts to the bioceramic preparation solution, and prepares bioceramic coprecipitated powder using a solution coprecipitation method. By adjusting the pH value and calcium / phosphorus ratio during the reaction process, bioceramic coprecipitated powders with different phases are prepared, and then the strontium / copper doped bioceramic microspheres are obtained.

5. The bone repair scaffold for promoting the repair of diabetic bone defects as described in claim 2, characterized in that, The method for preparing the porous polymer scaffold includes the following steps: S2 dissolves the polymer in an organic solvent and stirs thoroughly until clear; the strontium / copper doped bioceramic microspheres are added to the polymer solution and stirred thoroughly to obtain 3D printing ink, and the porous polymer scaffold is obtained by 3D printing.

6. The bone repair scaffold for promoting the repair of diabetic bone defects as described in claim 2, characterized in that, The method for preparing the scaffold body includes the following steps: S3 immerses the porous polymer scaffold in a coating solution containing PDGF-BB, repeatedly degassing under vacuum and drying to obtain the scaffold body with a PDGF-BB coating.

7. The bone repair scaffold for promoting bone defect repair in diabetic patients as described in claim 6, characterized in that: The concentration of the coating solution is 0.002-5%, the soaking time is 0.5-24h, the vacuum degree is 5-100Pa, and the drying temperature is 15℃-150℃.

8. The bone repair scaffold for promoting the repair of diabetic bone defects as described in claim 2, characterized in that: The porous polymer scaffold has a porosity of 30% to 90% and a pore size of 10 to 2000 μm.

9. The bone repair scaffold for promoting the repair of diabetic bone defects as described in claim 2, characterized in that: The thickness of the bio-coating is 0.005–0.4 mm.

10. The bone repair scaffold for promoting the repair of diabetic bone defects as described in claim 2, characterized in that: The weight ratio of bioceramics, polymers, and platelet-derived growth factors is 50–90: 10–50: 0.001–0.1.

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