Preparation method and application of tea polyphenol coating modified 3D printing strontium-doped hydroxyapatite composite scaffold

Abstract

The invention discloses a preparation method and application of a 3D printing strontium-doped hydroxyapatite composite scaffold modified by a tea polyphenol coating. According to the preparation method of the 3D printing strontium-doped hydroxyapatite composite scaffold modified by the tea polyphenol coating, fibroin is used as an ion deposition template; the fibroin / strontium modified nano-hydroxyapatite particles are synthesized in a'green 'manner in a biomimetic mineralization manner, pollution discharge is avoided, the method is simple and convenient, organic and inorganic combination can be realized, the structure of an extracellular matrix of bone cells can be simulated, so that the effect of higher bone regeneration potential is brought, and meanwhile, active elements are doped, so that angiogenesis activity is brought; the surface of the stent is modified by adopting a stable layer-by-layer self-assembly coating technology of polyelectrolyte, a stable coating and continuous drug release can be provided, the immune microenvironment can be effectively regulated and controlled due to the existence of EGCG in the coating, and a good environment is created for regeneration and repair of bone defects.

Description

Technical Field

[0001] The present invention relates to the technical field of composite materials, and particularly to a preparation method and application of a 3D printed strontium-doped hydroxyapatite composite scaffold modified with a tea polyphenol coating. Background Art

[0002] Hydroxyapatite (HA) is a bioactive ceramic with good bioactivity, biocompatibility, and osteoconductivity. More importantly, HA has the same chemical structure as the inorganic components of human bone tissue and can be absorbed and utilized by bone tissue. Therefore, it is widely used in the preparation of bone tissue engineering materials. Strontium (Sr) and calcium (Ca) have similar physical and chemical properties and are both essential trace elements involved in the bone mineralization process. They play an important role in regulating the structure and strength of human bones. In vitro and in vivo studies have shown that strontium-containing biomaterials can induce the osteogenic differentiation of mesenchymal stem cells while inhibiting the formation of osteoclasts. In addition, it has been found that strontium can promote endothelial cell angiogenesis and repair infarcted hearts, suggesting that strontium is a key element for promoting blood vessel development and osteogenic differentiation. Therefore, partial substitution of calcium in hydroxyapatite with strontium can produce a multifunctional composite material with excellent angiogenesis and osteogenic properties.

[0003] HA nanocrystals are the main component of the inorganic materials in the extracellular matrix of bone cells and, together with organic components including collagen and lipids, form the extracellular matrix of bone cells. Therefore, bone tissue engineering biomaterials composed of organic and inorganic substances can mimic the structure of the extracellular matrix of bone cells, thereby bringing higher bone regeneration potential. Many natural compounds have been used as templates for HA mineralization and are used to prepare bone tissue engineering biomaterials with osteogenic inductive activity. In recent years, the use of proteins as organic templates for the preparation of hydroxyapatite nanocrystals has received much attention because it effectively avoids the introduction of toxic and harmful chemical reagents. In addition, researchers have found that the process of HA nucleation begins with the binding of anionic side chains in proteins to calcium ions. Therefore, with the increase in proteins and acidic amino acids, the nucleation of HA is promoted. Silk fibroin (SF) is a natural macromolecular fibrous protein derived from silk, with good biodegradability and biocompatibility, and is widely used in biomedical fields such as drug delivery, wound dressings, artificial blood vessels, and bone tissue scaffolds. Interestingly, the combination of HA and SF can enhance biocompatibility, promote osteogenesis, and even reduce inflammation of aseptic implanted biomaterials in vivo. Therefore, we plan to select SF as an organic mineralization template for depositing strontium-substituted hydroxyapatite (Sr-HA) and use a chemical co-precipitation hydrothermal method to greenly and biomimetically prepare Sr-SF-HA particles.

[0004] However, local direct implantation of Sr-SF-HA particles may lead to rapid failure, and the rapid release of ions in a short period may cause more harm than benefit to tissues. Loading Sr-SF-HA particles into carriers such as 3D scaffolds, hydrogels, fibrous membranes, and microspheres can perfectly solve this problem and achieve slow release of functional substances. Among them, 3D printing technology can create a suitable spatial structure for cell growth and migration by manufacturing porous scaffolds that adapt to the shape of bone defects. Classical 3D printing scaffolds based on polymers such as polycaprolactone (PCL) exhibit good biocompatibility and good mechanical strength close to natural bone, thus showing significant advantages.

[0005] In recent years, some studies have shown that after the implantation of biomaterials, immune cells affect angiogenesis and osteogenesis by regulating the early inflammatory response on the surface of the implant. Therefore, the impact of the immune microenvironment on biomaterials cannot be ignored, and surface coating modification is a widely used and effective strategy to solve this problem. Surface coatings can directly mediate the interaction between biomaterials and surrounding cells and tissues, thereby affecting the repair and regeneration of bone tissue. Among them, the stable layer-by-layer self-assembly (LBL) coating technology of polyelectrolytes has been explored as a general and easy-to-use technology in the biomedical field. Epigallocatechin-3-gallate (EGCG) is the main component of green tea polyphenols. As a polyphenol, it has anti-inflammatory, antioxidant, and immune microenvironment regulation functions. It can also exhibit strong adhesion strength on the surface of various materials through oxidative self-polymerization in a weakly alkaline environment. In addition, studies have shown that PEI can crosslink with catechol through Michael addition and Schiff base reactions, and together with the π-π stacking between aromatic phenols and rich hydrogen bond forces, further enhance the stability of the coating. Therefore, EGCG and PEI are ideal raw material combinations for modifying the LBL coating on the surface of biomaterials to provide a stable coating and sustained drug release.

[0006] Clinically, maxillofacial bone defects often caused by trauma, tumors, hypoplasia, inflammation, surgical resection, etc. seriously affect the physical and mental health of patients. Traditionally, autologous bone grafting has been regarded as the gold standard for bone defect repair. However, this method has some problems, such as limited graft sources and prone to postoperative complications. In recent years, through the combination of new knowledge and new technologies in materials science, biology and medicine, bone tissue engineering has made great progress and is one of the most promising methods for repairing bone defects. Bone defect repair is a complex biological process involving the interaction between different complex biological events, including antioxidant, immunomodulatory, early angiogenesis and osteogenic differentiation. In the face of the body's own immunogenic response and various pathological bone defects such as diabetic bone defects, osteoporosis bone defects, and periodontitis bone defects, antioxidant and immunomodulatory capabilities can establish a microenvironment conducive to bone defect repair locally. In the initial stage of bone defect repair, new blood vessels can transport oxygen, nutrients and biological factors to the defect area, thus promoting bone tissue regeneration. Therefore, an ideal tissue engineering material for bone defect regeneration should simultaneously achieve immunomodulation, functional vascularization and bone regeneration.

[0007] In the prior art, in the invention patent with the application number CN202310356654.7, the publication date of December 8, 2023, and the title of "A 3D Printed Strontium-Doped Hydroxyapatite Composite Scaffold and Its Preparation Method", a preparation method of a strontium-doped calcium phosphate bone repair scaffold with a TPMS structure is disclosed. It is characterized by the following steps: Step 1. Prepare the printing slurry: Weigh a certain amount of calcium phosphate, strontium carbonate, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (TPO) and carbon black solid-phase powders, and mix them evenly with the liquid-phase components of KOS163 dispersant, 1,6-hexanediol diacrylate (HDDA) and trimethylolpropane triacrylate (TMPTA) to obtain the printing slurry of strontium-doped calcium phosphate; Step 2. 3D printing: Pre-design the three-dimensional model of the scaffold with a TPMS structure in computer software, import it into the printing device, pour the mixed slurry in Step 1, and use digital light processing (DLP) 3D printing technology for printing to obtain the rough scaffold product; Step 3: Degreasing and sintering: Clean and dry the rough scaffold product in Step 2, and sinter it step by step in a muffle furnace to finally obtain a strontium-doped calcium phosphate bone repair scaffold with a TPMS structure. However, this technical solution has some problems. The first point is that this solution uses a variety of organic polymerization raw materials and combines the preparation method of degreasing and sintering. The synthesis method will cause a large amount of pollution and is not conducive to implementation. Secondly, the biological function of the material synthesized by this solution is single and does not have the function of immunomodulation.

[0008] In the prior art, in the invention patent with the application number CN202210154641.7, the publication date of January 26, 2024, and the title of "A Strontium-Doped Nano-Hydroxyapatite Microsphere / Chitosan Hydrogel and Its Preparation Method", a strontium-doped nano-hydroxyapatite microsphere / chitosan hydrogel and its preparation method are disclosed. The hydroxyapatite microspheres are synthesized by microwave-assisted hydrothermal method for standby. A chitosan gel is made from an aqueous solution of KOH / urea and chitosan. The chitosan gel and the hydroxyapatite microspheres are mixed at a solid-liquid ratio of (0.05 - 0.2 g):10 mL, and stirred with a magnetic stirrer for 1 h to obtain a uniform milky white composite hydrogel, namely the strontium-doped nano-hydroxyapatite microsphere / chitosan hydrogel composite material. The prepared hydrogel effectively improves the osteogenic effect of traditional strontium-doped materials, and the preparation process and method are simple, and the injectability is good. However, the microspheres prepared by this technology have insufficient mechanical properties when facing large segmental bone defects in the maxillofacial region and cannot effectively restore the continuity of bone defects. Moreover, the synthesized hydrogel has a relatively single function and does not have the function of immune regulation.

[0009] Therefore, it has become a problem worthy of research to provide a preparation method and application of a 3D printed strontium-doped hydroxyapatite composite scaffold modified with a tea polyphenol coating doped with active elements with excellent morphological controllability, mechanical properties, biocompatibility, and simultaneously having immune regulation, promoting angiogenesis and osteogenesis functions. Summary of the Invention

[0010] The object of the present invention is to provide a preparation method and application of a 3D printed strontium-doped hydroxyapatite composite scaffold modified with a tea polyphenol coating doped with active elements with excellent morphological controllability, mechanical properties, biocompatibility, and simultaneously having immune regulation, promoting angiogenesis and osteogenesis functions.

[0011] The object of the present invention is achieved as follows:

[0012] A preparation method of a 3D printed strontium-doped hydroxyapatite composite scaffold modified with a tea polyphenol coating includes the following steps:

[0013] Step 1: Remove the sericin protein in the silkworm cocoon by high-temperature alkaline degumming method to obtain silk fibroin for standby;

[0014] Step 2: Dissolve the silk fibroin in step 1 in a 9.3 M lithium bromide aqueous solution to obtain a liquid, transfer the liquid to a dialysis bag, and dialyze the mixture with deionized water for 3 days to remove lithium bromide; obtain a silk fibroin solution and store it at 4°C;

[0015] Step 3. Use CaCl 2 、SrCl 2 、Na 3 PO 4The silk fibroin solution in Step 2 is used to synthesize Sr-SF-HA nanoparticles by hydrothermal co-precipitation method;

[0016] Step 4. Use a transmission electron microscope equipped with energy-dispersive spectroscopy (EDS) to characterize the morphology and elemental composition of the Sr-SF-HA nanoparticles obtained in Step 3;

[0017] Step 5. 3-6 g of the Sr-SF-HA nanoparticles obtained in Step 3 are evenly dispersed in 40-60 mL of dichloromethane, and then 10-20 g of PCL particles are added. After the PCL particles are completely melted, a uniform solution is prepared; and the uniform PCL / DCM solution is quickly poured into a glass dish and continuously stirred to evaporate DCM to form a PCL composite film uniformly containing Sr-SF-HA nanoparticles;

[0018] Step 6. Use software on a computer to design the parameters of the porous cylindrical scaffold; then load the PCL composite film raw material containing Sr-SF-HA nanoparticles in Step 5 into a high-temperature barrel and heat it to 120 °C; meanwhile, set the printing parameters. After the raw material is completely melted, printing is carried out to obtain a PCL / Sr scaffold;

[0019] Step 7. Use the method of layer-by-layer self-assembly to prepare an EGCG / PEI coating on the PCL / Sr scaffold; dissolve 2 mg / mL EGCG and 1 mg / mL PEI in Tris buffer to form an EGCG / PEI coating solution, and use a UV-visible spectrophotometer to measure the UV absorption peak of the coating solution; and immerse the PCL / Sr scaffold in the above EGCG / PEI coating solution for 4 hours; then, wash the PCL / Sr scaffold with deionized water and dry it to obtain an EP@PCL / Sr scaffold with an EGCG / PEI coating; the pH value of the Tris buffer is 8.5. The results are as Figure 6 shown.

[0020] Step 8. Characterize the morphology and elemental composition of the EP@PCL / Sr scaffold by a field emission scanning electron microscope equipped with an energy-dispersive spectrometer (EDS); the results are as Figure 7 、 Figure 8 shown.

[0021] Step 9. Experiments on the immunomodulatory ability, bone induction characteristics, and angiogenesis-promoting ability of the EP@PCL / Sr scaffold.

[0022] The specific operation of the above Step 1 is as follows: Put the silkworm cocoons into a 2% w / v sodium carbonate solution, soak them at 120 °C for 30 minutes, then wash them three times with deionized water to remove sericin, leaving silk fibroin, and place the silk fibroin in an oven to dry for later use.

[0023] The specific operations of step 3 are as follows: Step 3.1 Dissolve 2-3 g of CaCl 2 , 0.3-0.5 g of SrCl 2 in 40-50 mL of deionized water, then add 10 mL of 5% w / v silk fibroin solution and mix evenly; Step 3.2 Dissolve 5-6 g of Na 3 PO 4 and 0-1 g of NaOH in 50 mL of deionized water and mix evenly; Step 3.3 Gradually add the solution containing Na 3 PO 4 and NaOH dropwise to the silk fibroin solution containing SrCl 2 and CaCl 2 to obtain a suspension, and adjust the pH to 11; Stir the suspension at 60 °C for 24 hours for sufficient reaction, and age it at room temperature for 24 hours; Step 3.4 Filter the suspension to collect Sr-SF-HA nanoparticles, and wash them three times with deionized water; Step 3.5 Dry the Sr-SF-HA nanoparticles at 60 °C for 48 hours.

[0024] In step 4, X-ray diffraction (XRD) is used to evaluate the crystal phase and crystallinity of the particles; X-ray photoelectron spectroscopy (XPS) is used to evaluate the surface element composition; and Fourier transform infrared spectroscopy (FTIR) is used to characterize the chemical functional groups of the surface substances. The results are as Figures 2 - 4 shown

[0025] In step 6, the printer parameters are set as follows: Use a nozzle with a diameter of 0.2 mm, a filling spacing of 1 mm, a layer height of 0.25 mm, lay the pattern in a 0 / 90° rotation direction, set a printing speed of 4 mm / s and a material extrusion output speed of 0.1 mm / s; After the raw material is completely melted, perform printing. An application of a tea polyphenol-coated 3D printed strontium-doped hydroxyapatite composite scaffold, the application of a tea polyphenol-coated 3D printed strontium-doped hydroxyapatite composite scaffold in repairing bone defects.

[0026] The beneficial effects of the present invention are as follows: (1) The present invention uses silk fibroin as an ion deposition template to "greenly" synthesize silk fibroin / strontium-modified nano-hydroxyapatite particles through biomimetic mineralization, with no pollution emissions, simple and convenient, and can achieve the combination of organic and inorganic, simulating the structure of the extracellular matrix of bone cells, thus bringing the effect of higher bone regeneration potential; (2) The present invention uses 3D printing technology to prepare a composite scaffold, which can be customized and modeled for different bone defects by using clinical CT data, etc., and can best restore the continuity and shape of the bone defect; (3) The present invention uses the stable layer-by-layer self-assembly coating technology of polyelectrolytes to modify the surface of the scaffold, which can provide a stable coating and continuous drug release; (4) With the doping mineralization of metal ions and functional coatings, the composite scaffold is endowed with multiple biological functions such as scavenging reactive oxygen species, regulating macrophage polarization, promoting angiogenesis and osteogenic repair. The present invention breaks the functional singularity of biomaterials and has broad application prospects in the field of bone tissue engineering. Brief Description of the Drawings

[0027] Figure 1 Schematic diagram of the material preparation of EP@PCL / Sr of the present invention;

[0028] Figure 2 TEM image of Sr-SF-HA nanoparticles of the present invention;

[0029] Figure 3 EDS detection result diagram of Sr-SF-HA nanoparticles of the present invention;

[0030] Figure 4 XPS detection result diagram of Sr-SF-HA nanoparticles of the present invention;

[0031] Figure 5 XRD and FTIR detection result diagrams of Sr-SF-HA nanoparticles of the present invention;

[0032] Figure 6 UV spectrum detection of the coating modification solution and physical diagram of the scaffold of the present invention;

[0033] Figure 7 SEM images of the surface and cross-section of the EP@PCL / Sr scaffold of the present invention;

[0034] Figure 8 EDS detection result diagram of the EP@PCL / Sr scaffold of the present invention;

[0035] Figure 9 Anti-inflammatory ability detection result diagram of the EP@PCL / Sr scaffold of the present invention;

[0036] Figure 10 Osteogenic ability detection result diagram of the EP@PCL / Sr scaffold of the present invention;

[0037] Figure 11 This is the angiogenic ability detection result diagram of the EP@PCL / Sr scaffold of the present invention. Detailed implementation manners

[0038] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0039] The preparation method of the tea polyphenol-coated modified 3D printed strontium-doped hydroxyapatite composite scaffold is as Figure 1 shown and includes the following steps:

[0040] Step 1. First, remove the sericin in the cocoon by the high-temperature alkaline degumming method. Put the cocoon into a 2% (w / v) sodium carbonate solution, soak it at 120 °C for 30 minutes, then wash it three times with deionized water to remove the sericin, leaving the fibroin. Place the fibroin in an oven to dry for later use;

[0041] Step 2. Dissolve the fibroin in step 1 in a 9.3 M lithium bromide aqueous solution, then transfer the liquid to a dialysis bag and dialyze the mixture with deionized water for 3 days to remove the lithium bromide. Finally, a fibroin solution is obtained and stored at 4 °C.

[0042] Step 3. Use calcium chloride (CaCl 2 ), strontium chloride (SrCl 2 ), sodium phosphate (Na 3 PO 4 ) and the fibroin solution in step 2 to synthesize Sr-SF-HA nanoparticles by the hydrothermal co-precipitation method. The specific operations are as follows: Step 3.1 Dissolve 2.4975 g of CaCl 2 , 0.3963 g of SrCl 2 in 40 ml of deionized water, then add 10 ml of 5% w / v fibroin solution and mix evenly; Step 3.2 Dissolve 5.7 g of Na 3 PO 4 and 0.2 g of NaOH in 50 ml of deionized water and mix evenly; Step 3.3, dropwise add the solution containing Na 3 PO 4 and NaOH to the fibroin solution containing SrCl 2 and CaCl 2 to obtain a suspension, and adjust the pH to 11; stir the suspension at 60 °C for 24 hours for sufficient reaction and age it at room temperature for 24 hours; Step 3.4 Filter the suspension to collect the Sr-SF-HA nanoparticles and wash them three times with deionized water; Step 3.5, dry the Sr-SF-HA nanoparticles at 60 °C for 48 hours.

[0043] Step 4. A transmission electron microscope (TEM) equipped with energy-dispersive spectroscopy (EDS) was used to characterize the morphology and elemental composition of Sr-SF-HA particles. X-ray diffraction (XRD) was used to evaluate the crystal phase and crystallinity of the particles. The surface elemental composition was evaluated by X-ray photoelectron spectroscopy (XPS). The chemical functional groups of the surface substances were characterized by Fourier transform infrared spectroscopy (FTIR), and the results are as Figures 2 - 5 shown. It can be found by TEM detection that the Sr-SF-HA particles are nanorod-like structures. In addition, the characteristic lattice spacings of hydroxyapatite can be observed in the high-magnification images, including the spacings of 0.334 nm corresponding to the (002) plane and 0.272 nm corresponding to the (211) plane, respectively. By EDS detection, it can be found that the (Ca+Sr) / P ratio in Sr-SF-HA is 1.56, and the result is close to 1.67, which is almost the Ca / P ratio of natural hydroxyapatite. Through the full XPS spectrum, it can be observed that the Sr-SF-HA nanoparticles contain the elements Sr, Ca, N, P, O, and C. By studying the fine spectra, Sr3p and Sr3d are the two main peaks of strontium, and they are detected at 269.1 eV (Sr 3p3 / 2), 280.1 eV (Sr 3p1 / 2), 134 eV (Sr 3d3 / 2), and 132.8 eV (Sr 3d5 / 2). The peaks of C1s can be fitted at 284.6 eV, 285.9 eV, and 287.8 eV, corresponding to three main components, including the chemical bonds of C–C, C–O / C–N, and C=O / C=N, respectively. The X-ray diffraction (XRD) results show that both SF-HA and Sr-SF-HA have the characteristic diffraction peaks of hydroxyapatite crystals (JCPDS No. 09–0432) at (002), (210), (211), (300), (202), (310), (222), and (213) without any displacement or new peaks. These results indicate the successful synthesis of Sr-SF-HA nanoparticles.

[0044] Step 5. 3 g of the Sr-SF-HA nanoparticles obtained in Step 3 were uniformly dispersed in 40 mL of dichloromethane, and then 10 g of PCL particles were added. After the PCL particles were completely melted, a uniform solution was prepared; and the uniform PCL / DCM solution was quickly poured into a glass dish and continuously stirred to evaporate DCM to form a PCL composite film uniformly containing Sr-SF-HA nanoparticles.

[0045] Step 6. The parameters of the porous cylindrical bracket were designed using software on the computer. Subsequently, the PCL composite film raw material was loaded into the high-temperature barrel and heated to 120°C. At the same time, the printing parameters were set as follows: using a 0.2mm diameter nozzle, a 1mm filling spacing, a 0.25mm layer height, a 0 / 90° rotation direction for laying the pattern, a 4mm / s printing speed, and a 0.1mm / s material extrusion output speed. After the raw material is completely melted, printing is performed.

[0046] Step 7. Prepare EGCG / PEI (EP) coating on PCL / Sr using a stacking self-assembly method. 2 mg / mL EGCG and 1 mg / mL PEI were dissolved in Tris buffer (pH 8.5) to form an EGCG / PEI coating solution, and the UV absorption peak of the coating solution was measured using a UV-visible spectrophotometer. The PCL / Sr scaffold was immersed in the solution for 4 hours. Then, the scaffold was washed with deionized water and dried to obtain an EP@PCL / Sr scaffold with EGCG / PEI coating; the results are shown in FIG. Figure 6 As shown. Pure PEI is a colorless and transparent solution, pure EGCG is a light brown solution, and the EGCG / PEI solution becomes a yellow-green solution. The absorbance of the three solutions was measured using UV-visible spectroscopy to further explore the reaction between EGCG and PEI. The results showed that EGCG and EGCG / PEI solutions had a peak at 323nm, which is the absorption peak of the quinone structure, indicating that under weak alkaline conditions, the catechol in EGCG has been oxidized to a quinone structure. The peak at 450nm in the EGCG / PEI solution is generated by the cross-linking structure between the quinone and amine groups through Schiff base and Michael addition reactions. After preparing the EGCG / PEI solution, we will soak the printed PCL / Sr scaffold to form a coating. It can be seen that after the coating is modified with the EGCG / PEI solution, the surface color of the scaffold turns yellow, indicating that the coating is successfully loaded.

[0047] Step 8. The morphology and elemental composition of the EP@PCL / Sr scaffolds were characterized by field emission scanning electron microscopy equipped with energy dispersive spectroscopy (EDS). Figure 7 , Figure 8 As shown. In the high-magnification surface and cross-sectional images, obvious Sr-SF-HA particles can be seen inside the scaffold and the nanostructured coating material with a thickness of 1-2 μm on the surface of the scaffold. EDS MAPPING images show that C, N, O, Ca, Sr and P are evenly distributed in the EP@PCL / Sr scaffold, indicating the uniform coating of EGCG / PEI and the uniform doping of Sr-SF-HA particles.

[0048] Step 9. Conduct relevant experiments on the immunomodulatory ability of the EP@PCL / Sr scaffold, the osteoinductive properties of the scaffold, and the ability of the scaffold to promote angiogenesis.

[0049] To study the immunomodulatory effect of the scaffold, first, we used flow cytometry to identify macrophage phenotypes. First, macrophages were seeded into 6-well plates at a density of 1×10 5 cells per well. Next, the cells were induced with LPS (100 ng / mL) and IL-4 (20 ng / mL) for 48 hours respectively. Then, the original medium was removed, and the cells were further treated with the medium containing the extract of each scaffold for 48 hours. After that, the macrophages were resuspended in PBS. Then, the cells were incubated with the primary antibody in the dark for half an hour. Finally, the phenotypes of macrophages (CD86 corresponding to M1 type, CD206 corresponding to M2 type) were analyzed by flow cytometry. Cells without LPS and IL-4 were used as the negative control group, and cells with only LPS and IL-4 were used as the positive control group.

[0050] Secondly, we further detected the proteins in macrophages through immunofluorescence experiments. Briefly, we seeded 2×10 4Raw264.7 cells were used, and the original cell morphology was observed under a microscope and images were collected. To detect the regulatory effect of the material on M1 macrophages, cells in each group except the control group were treated with an LPS solution at a concentration of 100 ng / mL for 48 hours, and then the treated cells were observed and images were collected. The cells were washed with PBS. Next, the cells in each group were cultured with media containing different scaffold extracts for 48 hours. Cells without LPS addition were used as the negative control group, and cells with only LPS addition were used as the positive control group. After treatment, the cells were fixed with 4% paraformaldehyde solution for 30 minutes, permeabilized with 0.5% Triton X-100, and incubated with 5% goat serum for 1.5 hours. Then they were incubated with iNOS antibody (1:100) overnight. Next, the cells were incubated with the secondary antibody (1:200) for 1 hour. Finally, DAPI staining solution was added and incubated in the dark for 15 minutes. The expression of iNOS was detected using a fluorescence microscope, and semi-quantitative analysis of the fluorescence intensity was performed using ImageJ. To detect the regulatory effect of the material on M2 macrophages, cells in each group except the control group were treated with an IL-4 solution at a concentration of 20 ng / mL for 48 hours, and then the treated cells were observed and images were collected. The cells were washed with PBS. Next, the cells in each group were cultured with media containing different scaffold extracts for 48 hours. Cells without IL-4 addition were used as the negative control group, and cells with only IL-4 addition were used as the positive control group. After treatment, the cells were fixed with 4% paraformaldehyde solution for 30 minutes, permeabilized with 0.5% Triton X-100, and incubated with 5% goat serum for 1.5 hours. Then they were incubated with iNOS antibody (1:100) overnight. Next, the cells were incubated with the secondary antibody (1:200) for 1 hour. Finally, DAPI staining solution was added and incubated in the dark for 15 minutes. The expression of CD206 was detected using a fluorescence microscope, and semi-quantitative analysis of the fluorescence intensity was performed using ImageJ. The results are as Figure 9 shown. The EP@PCL / Sr scaffold can effectively induce macrophage polarization towards the M2 type, inhibit macrophage polarization towards the M1 type, and has excellent immunomodulatory functions.

[0051] To investigate the osteogenic differentiation-promoting effect of the scaffold, rBMSCs were first seeded into 24-well plates and cultured until 80% confluence. Next, the medium containing different scaffold extracts was used to replace the conventional medium. To accurately identify the osteoinductive properties of the scaffold, no other external osteoinductive factors such as β-glycerophosphate, vitamin C, and dexamethasone were added. On the 7th day, the cells were fixed with 4% paraformaldehyde for 20 minutes to fix the rBMSCs and stained with BCIP / NBT alkaline phosphatase chromogenic method. In addition, alizarin red staining was used to detect calcium nodules. After 21 days of culture, the cells were washed with PBS and fixed in 4% paraformaldehyde for 30 minutes. Next, the calcium nodules were stained with alizarin red staining solution and then observed with a stereomicroscope.

[0052] The expression of osteogenesis-related proteins in rBMSCs was further investigated by immunofluorescence. Briefly, the polylysine-treated cell slides were placed into 12-well plates and rinsed with the medium. After washing three times, the medium was aspirated for standby. Then, rBMSCs were seeded into the corresponding 12-well plates containing cell slides. Next, the medium containing different scaffold extracts was used to replace the conventional medium. On the 4th day, the cells were fixed with 4% paraformaldehyde for 30 minutes. Next, the cells were permeabilized with 0.5% Triton X-100 and incubated with 5% goat serum for 1.5 hours. Then, they were incubated overnight with OPN and RUNX2 antibodies (1:100) respectively. Next, the cells were incubated with the secondary antibody (1:200) for 1 hour. Finally, the cytoskeleton was stained with F-actin (1:200) and the nucleus was stained with DAPI. The expression of OPN and RUNX2 was detected using a fluorescence microscope, and semi-quantitative analysis of the fluorescence intensity was performed using ImageJ. The results were as Figure 10 shown; on the 7th day, the cells in the EP@PCL / Sr treatment group had deeper blue staining than those in the CON group, indicating that the cells in the EP@PCL / Sr treatment group had stronger ALP activity and early osteogenic activity. On the 21st day, a large number of calcium nodules were produced by the cells in the EP@PCL / Sr treatment group, while only a small number of calcium nodules were produced in the CON group. This indicates that the cells in the EP@PCL / Sr treatment group not only had stronger early osteogenic activity but also led to stronger osteogenic differentiation results. The expression levels of OPN and RUNX2 proteins in the cells of the EP@PCL / Sr treatment group were also significantly higher than those in the CON group and the PCL treatment group. In summary, the EP@PCL / Sr scaffold has excellent osteogenic differentiation-promoting ability.

[0053] To study the angiogenesis-promoting effect of the scaffold. For the cell migration experiment, cells were seeded in six-well plates and cultured until 90% confluence. Then, a layer of cells was scraped off in the center of the cells using the tip of a pipette. Next, after washing with PBS, the cells were cultured in low-serum medium (FBS content 0.5%) containing the scaffold for 48 hours. Cells cultured in low-serum medium without the scaffold were used as the control group. Optical images of each well were captured at 0 and 48 hours using an optical microscope.

[0054] For the tube formation experiment, the Matrigel was thawed overnight at 4°C. Next, the Matrigel was evenly added to 24-well plates and incubated at 37°C for 30 minutes. Subsequently, the cells were seeded into the corresponding wells and cultured using medium containing different scaffold extracts. After 12 hours of culture, the cells were observed under a microscope.

[0055] The expression of angiogenesis-related proteins in HUVECs was further studied by immunofluorescence. Briefly, polylysine-treated cell slides were placed in 12-well plates and rinsed with medium, washed three times, and then the medium was aspirated for later use. Then, HUVECs (5×10 3 cells / well) were seeded into the corresponding 12-well plates containing the cell slides. Next, medium containing different scaffold extracts was used instead of the conventional medium. On the 4th day, the cells were fixed with 4% paraformaldehyde for 30 minutes. Next, the cells were permeabilized with 0.5% Triton X-100 and incubated with 5% goat serum for 1.5 hours. Then, they were incubated overnight with VEGF antibody (1:100). Next, the cells were incubated with the secondary antibody (1:200) for 1 hour. Finally, the cytoskeleton was stained with F-actin (1:200) and the nucleus was stained with DAPI. The expression of VEGF was detected using a fluorescence microscope, and semi-quantitative analysis of the fluorescence intensity was performed using ImageJ, and the results were as Figure 11 shown. The cells in the EP@PCL / Sr treatment group had stronger migration ability, ability to promote tube formation, and could significantly increase the protein expression of VEGF in endothelial cells, indicating that the scaffold has excellent angiogenic activity.

[0056] Application of the above-mentioned tea polyphenol-coated modified 3D printed strontium-doped hydroxyapatite composite scaffold in the restoration of bone defects.

Claims

1. A method for preparing a 3D printed strontium-doped hydroxyapatite composite scaffold modified with a tea polyphenol coating, characterized in that: The following steps are involved: Step 1: removing sericin from the cocoon by high temperature alkaline degumming to obtain silk fibroin for use; Step 2: dissolving the silk fibroin in step 1 in a set amount of lithium bromide aqueous solution to obtain a liquid, transferring the liquid to a dialysis bag, and dialyzing the mixture with deionized water for 3 days to remove the lithium bromide; The silk fibroin solution was obtained and stored at 4°C for future use; Step 3. Using CaCl2, SrCl2, Na3PO4 and the silk fibroin solution in step 2, Sr-SF-HA nanoparticles were synthesized by hydrothermal coprecipitation method; Step 4. Using a transmission electron microscope equipped with energy dispersive spectroscopy (EDS) to characterize the morphology and elemental composition of the Sr-SF-HA nanoparticles obtained in step 3; Step 5. 3-6 g of Sr-SF-HA nanoparticles obtained in step 3 are uniformly dispersed in 40-60 ml of dichloromethane, and then 10-20 g of PCL particles are added to prepare a uniform solution after the PCL particles are completely melted; and the uniform PCL / DCM solution is quickly poured into a glass dish and continuously stirred to evaporate DCM to form a PCL composite film uniformly containing Sr-SF-HA nanoparticles; Step 6. Design the parameters of the porous cylindrical scaffold using software on a computer; then load the PCL composite film raw material containing Sr-SF-HA nanoparticles in step 5 into a high-temperature barrel and heat it to 120°C; at the same time, set the printing parameters, and after the raw material is completely melted, print to obtain a PCL / Sr scaffold; Step 7. Prepare EGCG / PEI coating on PCL / Sr scaffold using layer-by-layer self-assembly method; 2 mg / mL EGCG and 1 mg / mL PEI were dissolved in Tris buffer to form an EGCG / PEI coating solution, and the UV absorption peak of the coating solution was measured using a UV-visible spectrophotometer; and the PCL / Sr scaffold was immersed in the above EGCG / PEI coating solution for 4 hours; Then, the PCL / Sr scaffolds were washed with deionized water and dried to obtain the EP@PCL / Sr scaffolds with EGCG / PEI coatings; the pH value of the Tris buffer was 8.5; Step 8. Characterize the morphology and elemental composition of the EP@PCL / Sr scaffolds by field emission scanning electron microscopy equipped with energy dispersive spectrometer EDS; Step 9. Experiments on the immunomodulatory ability of the EP@PCL / Sr scaffold, the osteoinductive properties of the scaffold, and the ability of the scaffold to promote angiogenesis.

2. The method for preparing the tea polyphenol coating-modified 3D printed strontium-doped hydroxyapatite composite scaffold according to claim 1, characterized in that: The specific operation of step 1 is as follows: put the cocoons into a 2% w / v sodium carbonate solution, soak them at 120° C. for 30 minutes, then wash them three times with deionized water to remove the sericin and leave the silk fibroin, and then dry the silk fibroin in an oven for later use.

3. The method for preparing the tea polyphenol coating-modified 3D printed strontium-doped hydroxyapatite composite scaffold according to claim 1, characterized in that: The specific operation of step 3 is as follows: step 3.1, dissolving 2-3 g of CaCl2 and 0.3-0.5 g of SrCl2 in 40-50 ml of deionized water, and then adding 10 ml of 5% w / v silk solution and mixing evenly; step 3.2, dissolving 5-6 g of Na3PO4 and 0-1 g of NaOH in 50 ml of deionized water and mixing evenly; step 3.3, adding dropwise the solution containing Na3PO4 and NaOH to the silk solution containing SrCl2 and CaCl2 to obtain a suspension, and adjusting the pH to 11; stirring the suspension at 60°C for 24 hours for sufficient reaction, and aging at room temperature for 24 hours; step 3.4 filtering the suspension to collect Sr-SF-HA nanoparticles, and washing them three times with deionized water; step 3.5, drying the Sr-SF-HA nanoparticles at 60°C for 48 hours.

4. The method for preparing the tea polyphenol coating-modified 3D printed strontium-doped hydroxyapatite composite scaffold according to claim 1, characterized in that: In step 4, X-ray diffraction (XRD) is used to evaluate the crystalline phase and crystallinity of the particles; X-ray photoelectron spectroscopy (XPS) is used to evaluate the surface element composition; and Fourier transform infrared spectroscopy (FTIR) is used to characterize the chemical functional groups of the surface substances.

5. The method for preparing the tea polyphenol coating-modified 3D printed strontium-doped hydroxyapatite composite scaffold according to claim 1, characterized in that: In step 6, the printer parameters are set as follows: use a nozzle with a diameter of 0.2 mm, a filling spacing of 1 mm, a layer height of 0.25 mm, a 0 / 90° rotation direction for laying the pattern, set a printing speed of 4 mm / s and a material extrusion output speed of 0.1 mm / s; after the raw material is completely melted, printing is performed.

6. An application of a 3D printed strontium-doped hydroxyapatite composite scaffold modified with a tea polyphenol coating as claimed in any one of claims 1 to 5, characterized in that: Application of 3D printed strontium-doped hydroxyapatite composite scaffolds modified with tea polyphenols coating in repairing bone defects.

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