A polycaprolactone scaffold, its preparation method and application

Abstract

This invention belongs to the field of bone tissue engineering materials technology, and specifically relates to a polycaprolactone scaffold, its preparation method, and its applications. The polycaprolactone scaffold of this invention comprises a polycaprolactone scaffold body and a zeolite imidazole ester backbone-8 coating grown in situ on the surface of the polycaprolactone scaffold body. This invention achieves the goal of promoting bone regeneration and immune regulation by growing the zeolite imidazole ester backbone-8 coating in situ on the surface of the polycaprolactone scaffold body through a two-stage reaction. This results in the polycaprolactone scaffold exhibiting superior cell proliferation-promoting properties, cell adhesion-promoting properties, and macrophage polarization gene expression levels.

Description

Technical Field

[0001] This invention belongs to the field of bone tissue engineering materials technology, and specifically relates to a polycaprolactone scaffold, its preparation method and application. Background Technology

[0002] Ideal bone tissue engineering scaffolds are key tools for repairing large bone defects and promoting bone regeneration. Their core design requirement is a three-dimensional, interconnected, porous network structure that can simulate the natural extracellular matrix microenvironment of bone cells, supporting cell migration, nutrient delivery, angiogenesis, and complex intercellular interactions. 3D printing technology is a revolutionary manufacturing method that transforms digital three-dimensional models into physical objects. It constructs objects by layer-by-layer material deposition, eliminating the need for traditional molds or machining. Currently, the most commonly used 3D printing technologies include fused deposition modeling (FDM), selective laser sintering (SLS), and stereolithography. FDM is an additive manufacturing technology based on material extrusion. Its core principle involves using a computer-controlled hot melt nozzle to heat and melt thermoplastic filaments, which are then deposited layer by layer to form a three-dimensional solid structure. It can not only accurately replicate complex anatomical shapes but also create biomimetic bone structures that meet the needs of bone tissue engineering through programmable pore design and material composites, achieving precise, intelligent, and effective personalization in bone tissue engineering.

[0003] Recent research in bone immunology has revealed the crucial role of the immune microenvironment in bone repair. Macrophage phenotypic polarization is a key regulatory factor. Macrophage phenotypes can dynamically transform between M1 and M2 phenotypes. The M1 phenotype promotes an early inflammatory response to trauma, while the M2 phenotype promotes inflammation resolution and bone tissue repair by secreting anti-inflammatory factors. Therefore, exploring and designing scaffold materials capable of modulating macrophage polarization is expected to enhance the bone repair effects of biomaterials. Polycaprolactone, a semi-crystalline polymer, is widely used in tissue engineering due to its excellent biodegradability, biocompatibility, and mechanical properties. However, the inherent hydrophobic properties of pure polycaprolactone scaffolds significantly affect biological behaviors such as cell adhesion and proliferation, limiting its application in bone repair.

[0004] Currently, some studies have focused on constructing a zeolite imidazolium ester framework-8 (ZIF-8) coating on the surface of titanium implants using a biomimetic mineralization method to endow the scaffold with osteogenic activity; others have promoted vascularized bone regeneration by regulating angiogenesis-osteoogenesis coupling; still others have utilized ZIF-8 nanoparticles loaded with desferamine mesylate (DFO) and combined with sodium alginate (SA) hydrogel to construct an injectable SA / DFO@ZIF-8 composite system to promote vascularized bone regeneration; and some studies have used 3D printing technology to fabricate PLGA / β-TCP scaffolds and loaded indocyanine green (ICG) into ZIF-8 to prepare composite nanoparticles. These composite nanoparticles and total flavonoids (TF) from Drynaria fortunei were then combined using a double emulsion method to prepare composite microspheres. The PLGA / β-TCP scaffolds were then immersed in a chitosan solution containing the composite microspheres to prepare drug-loaded composite scaffolds for bone defect repair. However, the zeolite imidazole ester skeleton-8 / polycaprolactone scaffolds prepared by the above-mentioned existing technologies generally have the following problems: 1) the osteogenic activity of the scaffold depends on additional drugs; 2) it is difficult to achieve the effects of promoting bone regeneration and immune regulation at the same time; 3) the preparation process is complicated and difficult to industrialize. Summary of the Invention

[0005] The present invention aims to solve one or more technical problems existing in the prior art, and at least provide a beneficial alternative. Specifically, the present invention provides a polycaprolactone scaffold that can controllably release zinc ions, induce stem cell proliferation and osteogenic differentiation, and induce macrophage regeneration and differentiation polarization, thereby simultaneously achieving the effects of promoting bone regeneration and immune regulation; at the same time, it also has good hydrophilicity, mechanical strength and biocompatibility.

[0006] The inventive concept of this invention: The polycaprolactone scaffold of this invention includes a polycaprolactone scaffold body and a zeolite imidazole ester skeleton-8 coating grown in situ on the surface of the polycaprolactone scaffold body.

[0007] This invention involves in-situ growth of a zeolite imidazole ester framework-8 coating on the surface of a polycaprolactone scaffold. Zeolite imidazole ester framework-8 is a metal-organic framework material formed by the self-coordination of zinc ions with 2-methylimidazolium. It can slowly degrade in physiological environments, continuously releasing zinc ions (Zn). 2+ ), released Zn 2+ It has antibacterial effects and can induce macrophages to polarize to the M2 phenotype, inhibit the expression of inflammatory factors, and reduce inflammation at bone defect sites, thereby creating a microenvironment conducive to bone regeneration and achieving the purpose of promoting bone regeneration and immune regulation.

[0008] Therefore, a first aspect of the present invention provides a polycaprolactone scaffold.

[0009] Specifically, the polycaprolactone scaffold includes a polycaprolactone scaffold body and a zeolite imidazole ester backbone-8 coating grown in situ on the surface of the polycaprolactone scaffold body.

[0010] Preferably, the molecular weight of polycaprolactone in the polycaprolactone scaffold body is 48,000-80,000 Daltons.

[0011] Preferably, the thickness of the zeolite imidazole ester skeleton-8 coating is 0.05-5µm.

[0012] Preferably, the polycaprolactone includes at least one of ester-terminated polycaprolactone, hydroxyl-terminated polycaprolactone, carboxyl-terminated polycaprolactone, amino-terminated polycaprolactone, mercapto-terminated polycaprolactone, and aldehyde-terminated polycaprolactone.

[0013] A second aspect of the present invention provides a method for preparing the polycaprolactone scaffold described in the first aspect of the present invention.

[0014] Specifically, the preparation method of the polycaprolactone scaffold includes the following steps: (1) The polycaprolactone scaffold body was placed in an imidazole organic ligand solution, and then a zinc salt solution was added to carry out the first stage reaction to obtain scaffold 1; (2) The scaffold 1 is placed in an imidazole organic ligand solution, and then a zinc salt solution is added to carry out the second stage reaction to obtain the polycaprolactone scaffold. The concentration of the imidazole organic ligand solution in step (2) is greater than that in step (1); the concentration of the zinc salt solution in step (2) is greater than that in step (1).

[0015] This invention achieves in-situ growth of a zeolite imidazole ester backbone-8 coating on the surface of a polycaprolactone scaffold through two reactions, which enables the polycaprolactone scaffold to exhibit superior cell proliferation, immunomodulatory, and osteogenic induction properties.

[0016] Preferably, in step (1), the reaction temperature of the first stage reaction is 20-30℃ and the reaction time is 1-2h; for example, the temperature is 20℃, 25℃, 30℃, etc., and the time is 1h, 1.5h, 2h, etc.

[0017] Preferably, in step (1), the concentration of the imidazole organic ligand solution is 3.3-16.5 g / L; for example, 3.3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 16.5 g / L, etc.

[0018] Preferably, in step (1), the concentration of the zinc salt solution is 3-15 g / L; for example, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, etc.

[0019] Preferably, in step (1), the zinc salt solution is added at a rate of 0.01-0.02 mL / s.

[0020] Preferably, in step (1), the zinc salt solution is added during stirring, and the stirring speed is 400-600 rpm.

[0021] Specifically, in step (1), a thin and uniform initial coating is formed on the surface of the polycaprolactone matrix using a low concentration solution to improve the problem that the hydrophobic surface of the polycaprolactone matrix is ​​not conducive to the uniform growth of the crystal coating, and to provide uniform growth sites for the subsequent growth of the zeolite imidazole ester skeleton-8, so as to avoid uneven crystal distribution or agglomeration due to excessively fast reaction.

[0022] Preferably, in step (1), the mass-to-volume ratio of the polycaprolactone scaffold body to the 2-methylimidazole solution is 0.02-0.04 g / mL; for example, 0.02 g / mL, 0.03 g / mL, 0.04 g / mL, etc.

[0023] Preferably, in step (2), the reaction temperature of the second stage reaction is 25-35℃ and the reaction time is 1-2h; for example, the temperature is 25℃, 30℃, 35℃, etc., and the time is 1h, 1.5h, 2h, etc.

[0024] Preferably, in step (2), the concentration of the imidazole organic ligand solution is 17-33 g / L; for example, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L, 27 g / L, 28 g / L, 29 g / L, 30 g / L, 31 g / L, 32 g / L, 33 g / L, etc.

[0025] Preferably, in step (2), the concentration of the zinc salt solution is 15.5-30 g / L; for example, 15.5 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L, 27 g / L, 28 g / L, 29 g / L, 30 g / L, etc.

[0026] Preferably, in step (2), the zinc salt solution is added at a rate of 0.01-0.02 mL / s.

[0027] Preferably, in step (2), the zinc salt solution is added during stirring, and the stirring speed is 400-600 rpm.

[0028] Specifically, in step (2), based on the existing zeolite imidazole ester backbone-8 coating, a higher concentration of solution is used to promote the continued growth of the coating. While ensuring the uniformity of the coating, a thicker and more complete coating is constructed, which is beneficial for cell adhesion, proliferation, and stable Zn on the scaffold. 2+ release.

[0029] The two-stage in-situ growth method involves first applying a base coat to the surface of the substrate material. This allows the zeolite imidazole ester skeleton-8 to form a more uniform, dense, and robust coating on top of the first-stage coating during the second-stage reaction, thus enabling the full utilization of the bioactivity of the zeolite imidazole ester skeleton-8.

[0030] Preferably, in steps (1) and (2), the imidazole organic ligand solution comprises a 2-methylimidazolium solution.

[0031] Preferably, in steps (1) and (2), the zinc salt solution includes at least one of zinc nitrate solution, zinc sulfate solution, and zinc chloride solution.

[0032] Preferably, in steps (1) and (2), the imidazole organic ligand solution is an aqueous solution of imidazole organic ligand.

[0033] Preferably, in steps (1) and (2), the zinc salt solution is an aqueous solution of zinc salt.

[0034] Preferably, cleaning is performed after both the first stage reaction and the second stage reaction.

[0035] Preferably, the cleaning is performed using alternating water and alcohol.

[0036] Preferably, the water includes pure water.

[0037] Preferably, the alcohol includes methanol.

[0038] Preferably, the cleaning process after the first stage reaction is as follows: at a temperature of 20-25°C, first stir in water at 300-400 rpm for 5-10 minutes, then stir in alcohol at 300-400 rpm for 5-10 minutes, and repeat the water and alcohol washing process 2-4 times.

[0039] Preferably, after the first stage reaction and cleaning are completed, drying is carried out at a temperature of 40-50°C for a time of 25-35 minutes.

[0040] Specifically, after the first stage of the reaction, alternating washing with water and alcohol can remove unstable crystals and byproducts from the reaction process, thus eliminating interference from impurities.

[0041] Preferably, the cleaning process after the second stage reaction is as follows: at a temperature of 20-25°C, first stir in water at 100-200 rpm for 10-30 min, then stir in alcohol at 100-200 rpm for 10-30 min, and repeat the water and alcohol washing process 2-4 times.

[0042] Preferably, after the second stage reaction and cleaning are completed, drying is carried out at a temperature of 40-50°C for 8-12 hours.

[0043] Preferably, the polycaprolactone scaffold body is prepared by 3D printing.

[0044] Preferably, during 3D printing, the needle diameter is 0.4-0.6 mm.

[0045] Preferably, during the 3D printing process, the temperature of the barrel and the needle is 65-75°C.

[0046] Preferably, during 3D printing, the fiber spacing of the polycaprolactone fiber is (0.9-1.1) mm × (0.9-1.1) mm; for example, 1 mm × 1 mm, etc.

[0047] Preferably, the printing rate during 3D printing is 10-25 mm / s.

[0048] Preferably, during 3D printing, the fiber orientation of the polycaprolactone fiber is 0-90°.

[0049] Preferably, the air pressure during 3D printing is 100-150 kPa.

[0050] Preferably, during 3D printing, the support layer height is 0.2-0.25mm.

[0051] A third aspect of the present invention provides the application of the polycaprolactone scaffold described in the first aspect of the present invention in bone tissue repair and regeneration.

[0052] Compared with the prior art, the beneficial effects of the technical solution provided by the present invention are as follows: (1) The present invention grows a zeolite imidazole ester framework-8 coating in situ on the surface of the polycaprolactone scaffold body, and controls the release of Zn through the zeolite imidazole ester framework-8 (ZIF8). 2+ This achieves the goal of synergistic immune regulation to promote bone regeneration.

[0053] (2) This invention achieves in-situ growth of a zeolite imidazole ester backbone-8 coating on the surface of the polycaprolactone scaffold through a two-stage reaction. This results in superior cell proliferation, cell adhesion, macrophage polarization gene expression levels, immunomodulatory effects, and osteogenic induction properties of the polycaprolactone scaffold. Simultaneously, this invention employs an in-situ growth method, immersing polycaprolactone in a solution containing zinc nitrate and 2-methylimidazole to directly generate zeolite imidazole ester backbone-8 crystals on the polycaprolactone surface. The coating exhibits strong adhesion, uniformity, and controllable thickness, enhancing the hydrophilicity of the scaffold surface and facilitating cell adhesion. It also ensures the preservation of Zn. 2+ Stable release.

[0054] (3) Compared with direct incorporation of ZIF-8 for printing, this invention maximizes the bioactivity advantages of ZIF-8 because the ZIF-8 coating is concentrated on the scaffold surface and directly contacts the cells. This can improve the scaffold's cell proliferation, cell adhesion, macrophage polarization gene expression level, immunomodulatory and osteogenic induction properties. At the same time, it avoids the impact of particle blockage on the printing process caused by the incorporation of ZIF-8 into polycaprolactone during the preparation process.

[0055] (4) The raw materials of this invention have been industrialized, are readily available, and are inexpensive; the preparation process is simple, the equipment requirements are not high, and it is easy to industrialize. Attached Figure Description

[0056] Figure 1 These are scanning electron microscope (SEM) images of the polycaprolactone scaffold of Example 1 and the scaffolds of Comparative Examples 1-4 of the present invention. Figure 2 The porosity test results are shown in the figures for the polycaprolactone scaffolds of Examples 1-3 and the scaffolds of Comparative Examples 1-4 of the present invention. Figure 3 The graph shows the compressive strength test results of the polycaprolactone stents in Examples 1-3 and the stents in Comparative Examples 1-4 of the present invention. Figure 4 These are static images of the polycaprolactone scaffold in Example 1 and the scaffold droplets in Comparative Examples 1-4 of the present invention. Figure 5 The figures show the water contact angle results of the polycaprolactone scaffolds in Examples 1-3 and the scaffolds in Comparative Examples 1-4 of this invention. Figure 6 The images show the BMSCs cell proliferation results of the polycaprolactone scaffold in Example 1 and the scaffolds in Comparative Examples 1-4 of this invention. Figure 7 These are images showing the cell viability and mortality staining results of BMSCs on the polycaprolactone scaffold of Example 1 and the scaffolds of Comparative Examples 1-4 of the present invention. Figure 8 This is a graph showing the MPs polarization gene expression levels of the polycaprolactone scaffold in Example 1 and the scaffolds in Comparative Examples 1-4 of the present invention. Figure 9 These are ALP staining images from the in vitro osteogenicity test of the polycaprolactone scaffold in Example 1 and the scaffolds in Comparative Examples 1-4 of the present invention. Figure 10 This is a graph showing ALP activity in the in vitro osteogenicity test of the polycaprolactone scaffold in Example 1 and the scaffolds in Comparative Examples 1-4 of the present invention. Figure 11 The graph shows the expression of osteogenic genes ALP, COL1, and RUNX2 in the in vitro osteogenicity test of the polycaprolactone scaffold in Example 1 and the scaffolds in Comparative Examples 1-4 of this invention. Figure 12 This is a quantitative graph of calcium nodules in the in vitro osteogenicity test of the polycaprolactone scaffold in Example 1 and the scaffolds in Comparative Examples 1-4 of the present invention. Detailed Implementation

[0057] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0058] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0059] Example 1 This embodiment provides a polycaprolactone scaffold, which consists of a polycaprolactone scaffold body and a zeolite imidazole ester backbone-8 coating grown in situ on the surface of the polycaprolactone scaffold body. The polycaprolactone in the polycaprolactone scaffold body has a molecular weight of 48,000 Daltons and is ester-terminated polycaprolactone (DG-C100 from Jinan Daigang Biotechnology Co., Ltd.). The thickness of the zeolite imidazole ester backbone-8 coating is 3.5µm.

[0060] This embodiment also provides a method for preparing the above-mentioned polycaprolactone scaffold, the specific steps of which are as follows: (1) Place the fully dried polycaprolactone particles into the barrel of the 3D printer, match the needle, and set the printing parameters as follows: needle diameter 0.5mm, barrel and needle temperature 70℃, fiber spacing 1.0mm×1.0mm, printing speed 20mm / s, fiber orientation 0-90°, air pressure 100kPa, and scaffold layer height 0.2mm. Print layer by layer at room temperature to obtain the polycaprolactone scaffold body; (2) Dissolve 0.5g of 2-methylimidazole in 50mL of pure water and 0.225g of zinc nitrate hexahydrate in 25mL of pure water to obtain a 2-methylimidazole solution with a concentration of 10g / L and a zinc nitrate solution with a concentration of 9g / L. The polycaprolactone scaffold body obtained in step (1) is completely immersed in the 2-methylimidazole solution. During continuous magnetic stirring (400rpm), zinc nitrate solution is added dropwise at a rate of 0.01mL / s. The first stage of in-situ reaction is carried out at 20℃ for 1h. After the reaction is completed, the scaffold is stirred at 400rpm for 10min in pure water at 22℃. At the same temperature, it is stirred at 400rpm for 10min in methanol. The scaffold is washed with water and methanol alternately three times. After washing, it is dried at 45℃ for 30min to obtain scaffold 1. (3) Dissolve 1.25g of 2-methylimidazole in 50mL of pure water and 0.55g of zinc nitrate hexahydrate in 25mL of pure water to obtain a 2-methylimidazole solution with a concentration of 25g / L and a zinc nitrate solution with a concentration of 22g / L. Immerse the scaffold 1 obtained in step (2) completely in the 2-methylimidazole solution. During continuous magnetic stirring (400rpm), add zinc nitrate solution dropwise at a rate of 0.01mL / s. Perform the second stage in-situ reaction at 25℃ for 1h. Form a zeolite imidazole ester skeleton-8 coating on the surface of the scaffold through in-situ growth reaction. After the reaction, stir at 200rpm for 30min in pure water at 25℃. At the same temperature, stir at 200rpm for 30min in methanol. Wash with water and methanol alternately 3 times. After washing, dry at 50℃ for 12h to obtain a polycaprolactone scaffold with a zeolite imidazole ester skeleton-8 coating grown in situ on the surface.

[0061] Example 2 The difference between Example 2 and Example 1 is as follows: In Example 2, the printing needle diameter is 0.4 mm, the barrel and needle temperature is 75℃, the fiber spacing is 0.9 mm × 0.9 mm, the printing rate is 10 mm / s, the air pressure is 120 kPa, and the support layer height is 0.22 mm. In Example 2, the zinc salt used is zinc sulfate heptahydrate. During the first stage of in-situ reaction, the concentration of the 2-methylimidazole solution is 3.3 g / L, the concentration of the zinc salt solution (zinc sulfate solution) is 3 g / L, the reaction temperature is 30℃, the reaction time is 2 hours, and the zinc sulfate solution is added dropwise at a rate of 0.02 mL / s under magnetic stirring at 600 rpm. The first stage of cleaning is carried out at 25℃ in pure water. The mixture was stirred at 350 rpm for 7 min, then stirred at 350 rpm for 7 min in methanol. The mixture was washed three times alternately with water and methanol. The drying temperature was 50℃ for 35 min. In the second stage of the in-situ reaction, the concentration of the 2-methylimidazole solution was 17 g / L, the concentration of the zinc salt solution was 15.5 g / L, the reaction temperature was 35℃, and the time was 2 h. The zinc salt solution was added dropwise at a rate of 0.02 mL / s under magnetic stirring at 600 rpm. The second stage of washing was carried out at 20℃, with stirring at 100 rpm for 10 min in pure water, then stirring at 100 rpm for 10 min in methanol. The mixture was washed three times alternately with water and methanol. The drying temperature was 40℃ for 8 h.

[0062] The thickness of the zeolite imidazole ester backbone-8 coating obtained in Example 2 is 2.7µm.

[0063] Example 3 The difference between Example 3 and Example 1 is as follows: In Example 3, the diameter of the printing needle for the support frame is 0.6 mm, the temperature of the barrel and needle is 65℃, the fiber spacing is 1.1 mm × 1.1 mm, the printing rate is 25 mm / s, the air pressure is 150 kPa, and the support frame layer height is 0.25 mm; the zinc salt used in Example 3 is zinc chloride; in the first stage of the in-situ reaction in Example 3, the concentration of the 2-methylimidazole solution is 16.5 g / L, the concentration of the zinc salt solution (zinc chloride solution) is 15 g / L, the reaction temperature is 25℃, the time is 1.5 h, and the zinc salt solution is added dropwise at a rate of 0.015 mL / s under magnetic stirring at 500 rpm; the first stage of cleaning is carried out at 20℃ in pure water at a rate of 300... Stir at rpm for 5 min, then stir in methanol at 300 rpm for 5 min. Wash three times alternately with water and methanol. Dry at 40℃ for 25 min. In the second stage of in-situ reaction, the concentration of 2-methylimidazole solution is 33 g / L, and the concentration of zinc salt solution is 30 g / L. The temperature of the second stage of in-situ reaction is 30℃ and the time is 1.5 h. Under magnetic stirring at 500 rpm, zinc salt solution is added dropwise at a rate of 0.015 mL / s. The second stage of washing is carried out at 22℃, with stirring in pure water at 150 rpm for 20 min, then stirring in methanol at 150 rpm for 30 min. Wash three times alternately with water and methanol. Dry at 45℃ for 10 h.

[0064] The thickness of the zeolite imidazole ester backbone-8 coating obtained in Example 3 was 4.2 µm.

[0065] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the in-situ growth reaction of zeolite imidazole ester skeleton-8 in Comparative Example 1 only carried out the first stage reaction.

[0066] Specifically, the preparation method of the polycaprolactone scaffold in Comparative Example 1 is as follows: (1) Place the fully dried polycaprolactone granules into the barrel of the 3D printer, match the needle, and set the printing parameters as follows: needle diameter 0.5mm, barrel and needle temperature 70℃, fiber spacing 1.0mm×1.0mm, printing speed 10mm / s, fiber orientation 0-90°, air pressure 100kPa, scaffold layer height 0.2mm, print layer by layer at room temperature to obtain the polycaprolactone scaffold body; (2) Dissolve 0.5g of 2-methylimidazole in 50mL of pure water and 0.225g of zinc nitrate hexahydrate in 25mL of pure water to obtain a 2-methylimidazole solution with a concentration of 10g / L and a zinc nitrate solution with a concentration of 9g / L. Completely immerse the polycaprolactone scaffold body obtained in step (1) in the 2-methylimidazole solution. During continuous magnetic stirring (400rpm), add zinc nitrate solution dropwise at a rate of 0.01mL / s. Perform in-situ reaction at 20℃ for 1h. After the reaction is completed, stir at 400rpm for 10min in pure water at 25℃. At the same temperature, stir at 400rpm for 10min in methanol. Wash with water and methanol alternately three times. After washing, dry at 45℃ for 30min to obtain a polycaprolactone scaffold with a zeolite imidazole ester skeleton-8 coating grown in situ on the surface.

[0067] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the in-situ growth reaction of zeolite imidazole ester skeleton-8 in Comparative Example 2 only carried out the second stage reaction.

[0068] Specifically, the preparation method of the polycaprolactone scaffold in Comparative Example 2 is as follows: (1) Place the fully dried polycaprolactone granules into the barrel of the 3D printer, match the needle, and set the printing parameters as follows: needle diameter 0.5mm, barrel and needle temperature 70℃, fiber spacing 1.0mm×1.0mm, printing speed 20mm / s, fiber orientation 0-90°, air pressure 100kPa, and scaffold layer height 0.2mm. Print layer by layer at room temperature to obtain the polycaprolactone scaffold body. (2) Dissolve 1.25g of 2-methylimidazole in 50mL of pure water and 0.55g of zinc nitrate hexahydrate in 25mL of pure water to obtain a 2-methylimidazole solution with a concentration of 25g / L and a zinc nitrate solution with a concentration of 22g / L. Completely immerse the polycaprolactone scaffold body obtained in step (1) in the 2-methylimidazole solution. During continuous magnetic stirring (400rpm), add zinc nitrate solution dropwise at a rate of 0.01mL / s. Perform in-situ reaction at 25℃ for 1h to form a zeolite imidazole ester skeleton-8 coating on the scaffold surface through in-situ growth reaction. After the reaction, stir at 200rpm for 30min in pure water at 25℃. At the same temperature, stir at 200rpm for 30min in methanol. Wash with water and methanol alternately three times. After washing, dry at 50℃ for 12h to obtain a polycaprolactone scaffold with a zeolite imidazole ester skeleton-8 coating grown in situ on the surface.

[0069] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that the polycaprolactone scaffold in Comparative Example 3 does not contain the zeolite imidazole ester backbone-8 coating.

[0070] Specifically, the preparation method of the polycaprolactone scaffold in Comparative Example 3 is the same as step (1) in Example 1.

[0071] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that in Comparative Example 4, zeolite imidazole ester skeleton-8 and polycaprolactone were dissolved, blended, and then printed into a scaffold. Zeolite imidazole ester skeleton-8 was not used as a coating.

[0072] Specifically, the preparation method of the stent in Comparative Example 4 is as follows: (1) Dissolve 1.65 g of 2-methylimidazole in 50 mL of pure water and 0.75 g of zinc nitrate hexahydrate in 25 mL of pure water to obtain a 2-methylimidazole solution with a concentration of 33 g / L and a zinc nitrate solution with a concentration of 30 g / L. During continuous magnetic stirring (500 rpm), zinc nitrate solution was added dropwise to the 2-methylimidazole solution at a dropping rate of 0.02 mL / s. The reaction was carried out at 30 °C for 4 h. After the reaction was completed, the mixture was centrifuged at 10000 rpm for 15 min. The liquid was discarded, the substrate was retained, and the mixture was washed with water and methanol alternately three times. After washing, the mixture was dried at 45 °C for 8 h to obtain the zeolite imidazole ester skeleton-8. (2) Dissolve 2g of polycaprolactone (molecular weight of 45,000 Daltons) in 30mL of dichloromethane, add 0.02g of zeolite imidazole ester skeleton-8, and magnetically stir at 500rpm for 24h to obtain a uniformly dispersed zeolite imidazole ester skeleton-8 / polycaprolactone solution; in a fume hood, pour the zeolite imidazole ester skeleton-8 / polycaprolactone solution onto a glass petri dish (liquid thickness of 1mm), and place it under ventilation for 24h to obtain a zeolite imidazole ester skeleton-8 / polycaprolactone film; The zeolite imidazole ester backbone-8 / polycaprolactone film was placed in a 3D printing barrel and matched with a 0.5mm needle. The printing parameters were set as follows: barrel and needle temperature 80℃, fiber spacing 1.0mm×1.0mm, printing speed 10mm / s, fiber orientation 0-90°, air pressure 150kPa, and scaffold layer height 0.2mm. Layer-by-layer printing was started at room temperature to obtain the zeolite imidazole ester backbone-8 / polycaprolactone composite scaffold.

[0073] Performance testing 1. Scanning electron microscopy observation The surface morphology of the polycaprolactone scaffold of Example 1 and the scaffolds of Comparative Examples 1-4 was observed using a scanning electron microscope. The samples were freeze-dried and sputter-coated with gold before observation.

[0074] Scanning electron microscope (SEM) images of the polycaprolactone scaffold of Example 1 and the scaffolds of Comparative Examples 1-4 are shown below. Figure 1 As shown. Among them, Figure 1 The left-hand images in the figure show the fiber morphology of the scaffold in each group, with a scale bar of 300µm. Figure 1The middle images in the figure are the morphological images of the holes in the support for each group, and the scale bar is 300µm. Figure 1 The right-hand figures in the image show the surface morphology of the support for each group, with a scale bar of 100µm.

[0075] Depend on Figure 1 As can be seen, compared with Comparative Example 3, the pores on the surfaces of Comparative Examples 1, 2, and 4 are significantly reduced. Compared with Comparative Examples 1 and 2, the surface roughness of the scaffold in Example 1 is significantly increased, which can increase cell adhesion on the scaffold surface and thus be more conducive to cell proliferation. Meanwhile, the pore size of the scaffold in Example 1 is not significantly different from that of Comparative Examples 1, 2, 3, and 4. However, as shown in the scaffold surface morphology image on the right, the adhesion of zeolite imidazole ester backbone-8 particles in the scaffold of Example 1 is significantly increased.

[0076] 2. Porosity testing of the support structure The mass of the dried scaffold sample is recorded as M0. At room temperature, a specific gravity bottle is filled with anhydrous ethanol, and the total mass is recorded as M1. The sample is placed in the specific gravity bottle, and after sonication for 5 minutes to remove air bubbles, the total mass is measured and recorded as M2. The remaining mass of the sample is then measured and recorded as M3. The porosity of the scaffold is calculated using the formula: P = (M2 - M3 - M0) / (M1 - M3) × 100%.

[0077] The porosity test results of polycaprolactone scaffolds in Examples 1-3 and scaffolds in Comparative Examples 1-4 are as follows: Figure 2 As shown, due to the consistency of printing parameters maintained during the printing process, there was no significant difference in porosity among the different groups of supports.

[0078] 3. Stent mechanical strength test After drying, the support is placed in an INSTON testing machine (34sc). -5 On the support, the compression speed is 1 mm / min, and the mechanical properties of the support are tested.

[0079] The compressive strength test results of the polycaprolactone scaffolds in Examples 1-3 and the scaffolds in Comparative Examples 1-4 are as follows: Figure 3 As shown.

[0080] Depend on Figure 3 It can be seen that the zeolite imidazole ester skeleton-8 coating has little effect on the mechanical strength of the stent, and there is no significant difference in mechanical strength among the stent groups.

[0081] 4. Scaffold hydrophilicity test Place the support under test on the lifting platform, adjust the height of the lifting platform, and use a contact angle meter to drop a liquid onto the surface of the support. The liquid volume is 2μL each time. Adjust the position of the sample and the liquid drop to match the measurement baseline, take a picture to obtain a static image of the liquid drop at this time, and use contact angle measurement software to calculate the contact angle (θ) of each group of supports.

[0082] Static images of the droplets in the polycaprolactone scaffold of Example 1 and the scaffolds of Comparative Examples 1-4 are shown below. Figure 4 As shown. The water contact angles of the polycaprolactone scaffolds in Examples 1-3 and the scaffolds in Comparative Examples 1-4 are as follows. Figure 5 As shown.

[0083] Depend on Figure 4 , 5 It can be seen that the scaffold material in Comparative Example 3 exhibits strong hydrophobicity, with the largest water contact angle. The water contact angle of Comparative Examples 1-2, which incorporate zeolite imidazole ester backbone-8, is smaller than that of Comparative Example 3, while the water contact angle of the polycaprolactone scaffolds in Examples 1-3, prepared using a two-stage in-situ reaction, is significantly reduced. This indicates that the polycaprolactone scaffold prepared using the two-stage in-situ reaction of this invention has better hydrophilicity, and hydrophilicity is beneficial for cell proliferation and bone tissue regeneration.

[0084] 5. Scaffold cell proliferation promotion performance test After resuscitation, rat bone marrow mesenchymal stem cells (BMSCs) were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 μg / mL penicillin, and 100 μg / mL streptomycin at 37°C in a 5% CO2 incubator. Cells were passaged when confluence reached 70-80%, and all cells were passaged three times in this experiment. Before the experiment, scaffolds in each group were sterilized by cobalt-60 irradiation. The sterilized scaffold samples were placed in 48-well plates, and a blank control group without scaffolds was established. Each sample was seeded with 1 × 10⁶ cells. 4 Cells were cultured for 1, 3, and 7 days, after which the culture medium was removed, and cell proliferation activity on different scaffolds was assessed using a CCK-8 assay kit. Absorbance (OD value) was measured using a microplate reader.

[0085] The proliferation results of BMSCs cells in Example 1 (polycaprolactone scaffold) and Comparative Examples 1-4 are as follows: Figure 6 As shown. Figure 6 In the diagram, * indicates P < 0.05, ** indicates P < 0.01, and **** indicates P < 0.0001.

[0086] Depend on Figure 6 It can be seen that after one day of co-culture, Comparative Example 1 showed significantly more BMSCs proliferation than Comparative Example 2; the number of BMSCs proliferating in Example 1 was slightly higher than that in Comparative Example 2. However, after three and seven days of co-culture, the number of BMSCs proliferating in Example 1 was significantly higher than that in Comparative Examples 1-4 (p<0.05), indicating that the polycaprolactone scaffold of Example 1 can better induce stem cell proliferation.

[0087] 6. Scaffold cell adhesion promotion properties The polycaprolactone scaffold of Example 1, the scaffolds of Comparative Examples 1-4, and BMSCs were co-cultured for 3 days (the specific method is the same as the cell proliferation performance test above). The cell viability of BMSCs was determined by staining the cells on the sample surface with live-dead cell dye. Then, the morphology and distribution of cells on the sample surface were observed using an inverted fluorescence microscope.

[0088] The results of BMSC cell viability and death staining in Example 1 (polycaprolactone scaffold) and Comparative Examples 1-4 are as follows: Figure 7 As shown, the scale bars are all 500 μm.

[0089] Depend on Figure 7 It can be seen that the number of BMSCs adhering to the polycaprolactone scaffold in Example 1 was significantly greater than that in Comparative Examples 1-4, while the number of BMSCs adhering to the scaffold in Comparative Example 4 was very small. CCK-8 assay results showed that on day 7 of culture, the absorbance of Example 1 was significantly enhanced compared to Comparative Examples 1-4, indicating that the activity of BMSCs on the polycaprolactone scaffold in Example 1 was significantly higher than that in Comparative Examples 1-4.

[0090] 7. Detection of expression levels of scaffold-induced macrophage (MP) polarization genes The immune microenvironment surrounding the graft influences bone regeneration. This study systematically investigated the effects of different scaffolds on macrophage polarization and immunomodulatory properties by co-culturing mouse macrophages (RAW264.7 cells) with scaffolds and detecting the expression of macrophage polarization-related genes on days 1 and 3. The specific procedure is as follows: RAW264.7 cells were spaced at 5 × 10⁶ cells per well. 4 Cells were seeded at a density of 1000 cells per well on 48-well plates using a scaffold, with a blank control group (without scaffolds) included. On days 1 and 3, the culture medium was removed, and total RNA was extracted from each sample using a column extraction method. After successful RNA extraction, cDNA was obtained by reverse transcription using a PCR top heater. The expression of the M1 immunomarker genes tumor necrosis factor α (TNF-α) and recombinant human interleukin-1β (IL-1β) and the M2 immunomarker genes CD206 and arginase (ARG) in MPs was detected using real-time quantitative PCR, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used as an internal control gene.

[0091] The expression levels of MPs polarization genes in Example 1 (polycaprolactone scaffold) and Comparative Examples 1-4 scaffolds are as follows: Figure 8 As shown. Figure 8 In the diagram, * indicates P < 0.05, ** indicates P < 0.01, and **** indicates P < 0.0001.

[0092] Depend on Figure 8As can be seen, compared with Comparative Examples 1-4, Example 1 significantly upregulated the expression of M2 macrophage-related genes (CD206 and ARG) and inhibited the expression of M1 macrophage-related genes (TNF-α and IL-1β), promoting macrophage polarization towards the M2 type with anti-inflammatory and tissue repair functions, thereby helping to establish an anti-inflammatory microenvironment, achieve immune regulation, and promote bone regeneration.

[0093] 8. Evaluation of in vitro osteogenic properties The early osteogenic differentiation of stem cells (BMSCs) on different scaffold surfaces was determined by alkaline phosphatase (ALP) staining and activity assay. Specifically, 1×10 5 BMSCs were added to 48-well plates containing scaffold samples and blank wells. After culturing for 24 h, the supernatant was aspirated, and osteogenic induction culture medium (100 mL complete medium (10% fetal bovine serum, 100 μg / mL penicillin and 100 μg / mL streptomycin in DMEM medium) + 0.39 mg dexamethasone + 1.76 mg vitamin C + 306.11 mg β-glycerophosphate sodium) was added respectively, and the medium was changed regularly. After co-culturing for 7 and 14 days, the medium was aspirated, and the total RNA of the cells was extracted and tested with an ELISA reader to calculate its concentration. Subsequently, cDNA was obtained by reverse transcription, and the expression of ALP, runt-related transcription factor 2 (RUNX2), and type I collagen (COLI) was detected by real-time quantitative PCR, with GAPDH as an internal reference gene.

[0094] On day 7 of osteogenic induction, the scaffolds were stained with ALP using an ALP staining kit, and the ALP on the scaffolds was quantified using an alkaline phosphatase assay kit and the quinoline carboxylic acid method (BCA method). On day 14 of osteogenic induction, the scaffolds were stained with alizarin red staining solution, and the deposition of calcium nodules on the scaffolds was observed under a microscope. After observation, the calcium nodules were eluted with 10% hexadecylpyridine chloride (CPC) aqueous solution, and the expression of calcium nodules in each sample was quantified using an ELISA reader (OD=562nm).

[0095] The gene primer sequences used for in vitro osteogenic performance evaluation are shown in Table 1.

[0096] Table 1: Gene primer sequences used for in vitro osteogenic performance evaluation

[0097] ALP staining images during in vitro osteogenic performance testing of the polycaprolactone scaffold in Example 1 and Comparative Examples 1-4 are shown below. Figure 9 As shown; ALP activity graph as shown Figure 10 As shown; the expression of osteogenic genes ALP, COL1, and RUNX2 is as follows Figure 11 As shown; quantitative analysis of calcium nodules is as follows: Figure 12 As shown. Figure 10-12 In the diagram, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0098] Depend on Figure 9-12 It can be seen that, during staining on day 7, the Comparative Example 3 scaffold (pure PCL scaffold) did not significantly promote osteogenic differentiation of cells, and ALP staining was less and lighter in color. Compared to the Comparative Example 3 scaffold, the Comparative Example 1 scaffold, which underwent only the first stage of in-situ reaction to prepare the zeolite imidazole ester framework-8 coating, showed a slight increase in ALP staining, but the difference was not significant. However, the ALP staining was more pronounced in the Example 1 scaffold, which underwent two in-situ growth processes to prepare the coating. Simultaneously, the absorbance of calcium nodules in Example 1 was significantly higher than that in Comparative Examples 1-4, with a statistically significant difference (p<0.001).

[0099] Furthermore, the expression of osteogenic-related genes (ALP, COLI, and RUNX2) was detected on days 7 and 14 to assess the osteogenic differentiation trend of BMSCs treated with each scaffold. Compared with other groups, Example 1 showed significantly upregulated expression of osteogenic-related genes at all time points (p<0.05). This demonstrates that the osteogenic induction of the polycaprolactone scaffold in Example 1 was significantly superior to that of Comparative Examples 1, 2, 3, and 4.

[0100] In summary, this invention achieves the goal of promoting bone regeneration and immune regulation by growing a zeolite imidazole ester backbone-8 coating in situ on the surface of the polycaprolactone scaffold through a two-stage reaction. This results in the polycaprolactone scaffold exhibiting superior cell proliferation, cell adhesion, macrophage polarization gene expression levels, and osteogenic induction properties.

[0101] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A polycaprolactone scaffold, characterized in that, The polycaprolactone scaffold includes a polycaprolactone scaffold body and a zeolite imidazole ester backbone-8 coating grown in situ on the surface of the polycaprolactone scaffold body.

2. The polycaprolactone scaffold according to claim 1, characterized in that, The polycaprolactone in the polycaprolactone scaffold body has a molecular weight of 48,000-80,000 Daltons; and / or, the thickness of the zeolite imidazole ester backbone-8 coating is 0.05-5 µm.

3. The polycaprolactone scaffold according to claim 2, characterized in that, The polycaprolactone includes at least one of ester-terminated polycaprolactone, hydroxyl-terminated polycaprolactone, carboxyl-terminated polycaprolactone, amino-terminated polycaprolactone, mercapto-terminated polycaprolactone, and aldehyde-terminated polycaprolactone.

4. The method for preparing the polycaprolactone scaffold according to any one of claims 1-3, characterized in that, The preparation method includes the following steps: (1) The polycaprolactone scaffold body was placed in an imidazole organic ligand solution, and then a zinc salt solution was added to carry out the first stage reaction to obtain scaffold 1; (2) The scaffold 1 is placed in an imidazole organic ligand solution, and then a zinc salt solution is added to carry out the second stage reaction to obtain the polycaprolactone scaffold. The concentration of the imidazole organic ligand solution in step (2) is greater than that in step (1); the concentration of the zinc salt solution in step (2) is greater than that in step (1).

5. The preparation method according to claim 4, characterized in that, In step (1), the reaction temperature of the first stage reaction is 20-30℃, and the reaction time is 1-2h; And / or, in step (1), the concentration of the imidazole organic ligand solution is 3.3-16.5 g / L; And / or, in step (1), the concentration of the zinc salt solution is 3-15 g / L.

6. The preparation method according to claim 4, characterized in that, In step (2), the reaction temperature of the second stage reaction is 25-35℃ and the reaction time is 1-2h.

7. The preparation method according to claim 4, characterized in that, In step (2), the concentration of the imidazole organic ligand solution is 17-33 g / L; And / or, in step (2), the concentration of the zinc salt solution is 15.5-30 g / L.

8. The preparation method according to claim 4, characterized in that, In steps (1) and (2), the imidazole organic ligand solution includes a 2-methylimidazolium solution; And / or, in steps (1) and (2), the zinc salt solution includes at least one of zinc nitrate solution, zinc sulfate solution, and zinc chloride solution; And / or, washing is performed after both the first stage reaction and the second stage reaction are completed.

9. The preparation method according to claim 4, characterized in that, The polycaprolactone scaffold body was prepared by 3D printing.

10. The application of the polycaprolactone scaffold according to any one of claims 1-3 in bone tissue repair and regeneration.

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