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

Abstract

The present invention discloses a preparation method and application of a 3D-printed strontium-doped hydroxyapatite composite scaffold modified with a tea polyphenol coating. The preparation method of the 3D-printed strontium-doped hydroxyapatite composite scaffold modified with a tea polyphenol coating of the present invention uses silk fibroin as an ion deposition template and "green" synthesizes silk fibroin / strontium-modified nano-hydroxyapatite particles by biomimetic mineralization. The method has no pollution emissions, is simple and convenient, and can achieve organic and inorganic combination, simulate the structure of the extracellular matrix of bone cells, thereby bringing about a higher bone regeneration potential effect. At the same time, active elements are doped to bring about angiogenesis activity. The present invention adopts a stable layer-by-layer self-assembly coating technology of polyelectrolyte to modify the surface of the scaffold, which can provide a stable coating and continuous drug release. The presence of EGCG in the coating can effectively regulate the immune microenvironment and create a good environment for the regeneration and repair of bone defects.

Description

Technical Field

[0001] The present invention relates to the technical field of composite materials, and in particular 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 excellent 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, studies have found that strontium can promote endothelial cell angiogenesis and repair infarcted hearts, suggesting that strontium is a key element in promoting vascular development and osteogenic differentiation. Therefore, partial replacement of calcium in hydroxyapatite with strontium can produce a multifunctional composite material with excellent angiogenic and osteogenic properties.

[0003] HA nanocrystals are the primary inorganic component of the extracellular matrix (ECM) of bone, forming the ECM along with organic components including collagen and lipids. Therefore, bone tissue engineering biomaterials composed of organic and inorganic materials can mimic the structure of the ECM, thereby enhancing bone regeneration potential. Many natural compounds have been used as templates for HA mineralization and to prepare osteogenic biomaterials for bone tissue engineering. In recent years, the use of proteins as organic templates for the preparation of hydroxyapatite nanocrystals has garnered considerable attention due to their ability to avoid the introduction of toxic and hazardous chemical reagents. Furthermore, researchers have found that the nucleation process of HA begins with the binding of anionic side chains within the protein to calcium ions. Therefore, the addition of protein and acidic amino acids promotes HA nucleation. Silk fibroin (SF), a naturally occurring macromolecular fibrous protein derived from silk, exhibits excellent biodegradability and biocompatibility and is widely used in biomedical applications 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 following sterile implantation of biomaterials in vivo. Therefore, we planned to select SF as the organic mineralization template for depositing strontium-substituted hydroxyapatite (Sr-HA) and use a chemical co-precipitation hydrothermal method to prepare Sr-SF-HA particles in a green biomimetic manner.

[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 of time may cause more damage to the tissue than benefits. Loading Sr-SF-HA particles into carriers such as 3D scaffolds, hydrogels, fiber membranes and microspheres can perfectly solve this problem and achieve the slow release of functional substances. Among them, 3D printing technology can create a suitable spatial structure for cell growth and migration because it can produce porous scaffolds that adapt to the shape of bone defects, while classic polymer-based 3D printed scaffolds such as polycaprolactone (PCL) show good biocompatibility and good mechanical strength close to natural bones, thus showing significant advantages.

[0005] In recent years, several studies have demonstrated that after biomaterial implantation, immune cells influence angiogenesis and osteogenesis by modulating early inflammatory responses on the implant surface. Therefore, the influence of the immune microenvironment on biomaterials cannot be ignored, and surface coating modification is a widely used and effective strategy to address this issue. Surface coatings can directly mediate interactions between biomaterials and surrounding cells and tissues, thereby influencing bone repair and regeneration. Among these, stable layer-by-layer self-assembly (LBL) coatings of polyelectrolytes have been explored as a versatile and accessible technique in the biomedical field. Epigallocatechin-3-gallate (EGCG), a major component of green tea polyphenols, exhibits anti-inflammatory, antioxidant, and immune microenvironment-modulating properties. It also exhibits strong adhesion to various surfaces through oxidative self-polymerization in a weakly alkaline environment. Furthermore, studies have shown that PEI can crosslink with catechol via Michael addition and Schiff base reactions. The π-π stacking and abundant hydrogen bonding between aromatic phenols further enhance the stability of the coating. Therefore, EGCG and PEI are an ideal raw material combination for modifying LBL coating on biomaterial surfaces to provide stable coating and sustained drug release.

[0006] Clinically, maxillofacial bone defects, often caused by trauma, tumors, hypoplasia, inflammation, and surgical resection, severely impact patients' physical and mental health. Traditionally, autologous bone transplantation has been considered the gold standard for bone defect repair. However, this approach presents several challenges, including limited graft sources and the susceptibility to postoperative complications. In recent years, by integrating new knowledge and technologies from materials science, biology, and medicine, bone tissue engineering has made tremendous progress and is one of the most promising approaches for repairing bone defects. Bone defect repair is a complex biological process involving the interplay of diverse and complex biological events, including antioxidant activity, immune regulation, early angiogenesis, and osteoblastic differentiation. In the face of the body's own immunogenic responses and various pathological bone defects, such as diabetic bone defects, osteoporotic bone defects, and periodontal bone defects, antioxidant and immune regulatory abilities can enable the establishment of a local microenvironment conducive to bone defect repair. During the initial stages of bone defect repair, newly formed blood vessels transport oxygen, nutrients, and biological factors to the defect area, thereby 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, the invention patent with application number CN202310356654.7 and publication date of December 8, 2023, entitled “A 3D printed strontium-doped hydroxyapatite composite scaffold and its preparation method” discloses a method for preparing a strontium-doped calcium phosphate bone repair scaffold with a TPMS structure, which is characterized by comprising the following steps: Step 1. Prepare printing slurry: weigh a certain amount of calcium phosphate, strontium carbonate, diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide (TPO) and carbon black solid phase powder, and mix with KOS163 dispersant, 1,6-hexanediol diacrylate The liquid components of HDDA and trimethylolpropane triacrylate (TMPTA) are uniformly mixed to obtain a strontium-doped calcium phosphate printing slurry. Step 2. 3D printing: A 3D model of a scaffold with a TPMS structure is pre-designed in computer software and imported into a printing device. The mixed slurry from Step 1 is poured into the scaffold and printed using digital light processing (DLP) 3D printing technology to obtain a crude scaffold product. Step 3: Debinding and sintering: The crude scaffold product from Step 2 is cleaned and dried, and then sintered in a muffle furnace at gradually increasing temperatures to obtain a strontium-doped calcium phosphate bone repair scaffold with a TPMS structure. However, this technical solution has some problems. First, it utilizes multiple organic polymer raw materials and combines a debinding and sintering preparation method, which can cause a lot of pollution and is not conducive to implementation. Second, the material synthesized by this solution has a single biological function and does not have immune regulation.

[0008] Prior art, patent application number CN202210154641.7, published on January 26, 2024, and entitled "A Strontium-Doped Nanohydroxyapatite Microsphere / Chitosan Hydrogel and Preparation Method," discloses a strontium-doped nanohydroxyapatite microsphere / chitosan hydrogel and its preparation method. Hydroxyapatite microspheres are synthesized using a microwave-assisted hydrothermal method. Chitosan gel is prepared using a KOH / urea aqueous solution and chitosan. The chitosan gel and hydroxyapatite microspheres are mixed at a solid-liquid ratio of (0.05-0.2 g):10 mL and stirred in a magnetic stirrer for 1 hour to obtain a uniform, milky-white composite hydrogel. This hydrogel effectively improves the osteogenic efficacy of traditional strontium-doped materials, features a simple preparation process, and offers excellent injectability. However, the microspheres prepared by this technology have insufficient mechanical properties when used to repair large maxillofacial bone defects and cannot effectively restore the continuity of bone defects. In addition, the synthesized hydrogels have relatively simple functions and lack immunomodulatory effects.

[0009] Therefore, it is a problem worthy of study 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 having excellent morphological controllability, mechanical properties, biocompatibility, and immunomodulatory, angiogenic and osteogenic functions. Summary of the Invention

[0010] The purpose 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 having excellent morphological controllability, mechanical properties, biocompatibility, and immunomodulatory, angiogenic and osteogenic functions.

[0011] The object of the present invention is achieved like this:

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

[0013] Step 1: Remove the sericin in the cocoon through high temperature alkaline degumming method to obtain silk fibroin for use;

[0014] Step 2: Dissolve the silk fibroin obtained 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 against deionized water for 3 days to remove the lithium bromide; obtain a silk fibroin solution, and store it at 4°C;

[0015] Step 3. Synthesize Sr-SF-HA nanoparticles by hydrothermal coprecipitation using CaCl2, SrCl2, Na3PO4 and the silk fibroin solution in step 2;

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

[0017] Step 5. Disperse 3-6 g of the Sr-SF-HA nanoparticles obtained in step 3 evenly in 40-60 ml of dichloromethane, then add 10-20 g of PCL particles. After the PCL particles are completely dissolved, a uniform solution is prepared. The uniform PCL / DCM solution is quickly poured into a glass dish and continuously stirred to evaporate the DCM, thereby forming a PCL composite film uniformly containing the 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 membrane containing Sr-SF-HA nanoparticles from Step 5 into a high-temperature barrel and heat it to 120°C. Simultaneously, set the printing parameters, and after the raw material is completely melted, print it to obtain the PCL / Sr scaffold.

[0019] Step 7. Prepare EGCG / PEI coating on PCL / Sr scaffold using stacking self-assembly method; 2 mg / mL EGCG and 1 mg / mL PEI were dissolved in Tris buffer to form EGCG / PEI coating solution, and the UV absorption peak of the coating solution was measured using UV-visible spectrophotometer; and the PCL / Sr scaffold was immersed in the above EGCG / PEI coating solution for 4 hours; then, the PCL / Sr scaffold was washed with deionized water and dried to obtain EP@PCL / Sr scaffold with EGCG / PEI coating; the pH value of Tris buffer was 8.5. The results are shown in Figure 7. Figure 6 shown.

[0020] Step 8. The morphology and elemental composition of the EP@PCL / Sr scaffold were characterized by field emission scanning electron microscopy equipped with energy dispersive spectroscopy (EDS). Figure 7 、 Figure 8 shown.

[0021] 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.

[0022] The specific operation of step 1 is as follows: placing the silk cocoons in a 2% w / v sodium carbonate solution at 120° C. and soaking for 30 minutes, then washing them three times with deionized water to remove the sericin and leave the silk fibroin, and then drying the silk fibroin in an oven for later use.

[0023] 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.

[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 elemental composition; and Fourier transform infrared spectroscopy (FTIR) is used to characterize the chemical functional groups of the surface substances. Figure 2-Figure 4 shown

[0025] In step 6, the printer parameters are set as follows: a 0.2mm diameter nozzle, a 1mm fill spacing, a 0.25mm layer height, a 0 / 90° rotation for the pattern, a 4mm / s print speed, and a 0.1mm / s material extrusion output speed; printing is performed after the raw material is completely melted. A 3D-printed strontium-doped hydroxyapatite composite scaffold modified with a tea polyphenol coating is used for bone defect repair.

[0026] The beneficial effects of the present invention are as follows: (1) The present invention uses silk fibroin as an ion deposition template and synthesizes silk fibroin / strontium modified nanohydroxyapatite particles in a "green" manner through biomimetic mineralization, which has no pollution emissions, is simple and convenient, and can achieve organic and inorganic combination, simulate the structure of the extracellular matrix of bone cells, thereby bringing about a higher bone regeneration potential effect; (2) The present invention uses 3D printing technology to prepare composite scaffolds, and can use clinical CT data and other data to perform customized modeling and printing for different bone defects, which can restore the continuity and appearance of bone defects to the best extent; (3) The present invention uses a stable layer-by-layer self-assembly coating technology of polyelectrolytes to modify the scaffold surface, which can provide a stable coating and continuous drug release; (4) With the doping mineralization and functional coating of metal ions, the composite scaffold is endowed with multiple biological functions such as scavenging reactive oxygen species, regulating macrophage polarization, promoting angiogenesis and osteogenesis repair. The present invention breaks the functional singleness 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 This is the EDS test result of the Sr-SF-HA nanoparticles of the present invention;

[0030] Figure 4 This is the XPS test result of the Sr-SF-HA nanoparticles of the present invention;

[0031] Figure 5 Graph showing the XRD and FTIR test results of the Sr-SF-HA nanoparticles of the present invention;

[0032] Figure 6 The UV spectrum test of the coating modification solution of the present invention and the actual image of the stent;

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

[0034] Figure 8 This is the EDS test result of the EP@PCL / Sr scaffold of the present invention;

[0035] Figure 9 This is a graph showing the anti-inflammatory ability test results of the EP@PCL / Sr scaffold of the present invention;

[0036] Figure 10 This is a graph showing the osteogenic ability test results of the EP@PCL / Sr scaffold of the present invention;

[0037] Figure 11 This is a graph showing the angiogenesis ability test results of the EP@PCL / Sr scaffold of the present invention. DETAILED DESCRIPTION

[0038] The present invention will be further described below with reference to the accompanying drawings and examples.

[0039] Preparation method of 3D printed strontium-doped hydroxyapatite composite scaffold modified by tea polyphenol coating, such as Figure 1 As shown, the following steps are included:

[0040] Step 1. First, the sericin in the cocoons is removed by high-temperature alkaline degumming. The cocoons are soaked in a 2% (w / v) sodium carbonate solution at 120°C for 30 minutes. The cocoons are then washed three times with deionized water to remove the sericin and leave the fibroin. The fibroin is then dried in an oven for later use.

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

[0042] Step 3. Use calcium chloride (CaCl2), strontium chloride (SrCl2), sodium phosphate (Na3PO4) and the silk fibroin solution in step 2 to synthesize Sr-SF-HA nanoparticles by hydrothermal co-precipitation method. The specific operations are as follows: Step 3.1 Dissolve 2.4975 g of CaCl2 and 0.3963 g of SrCl2 in 40 ml of deionized water, then add 10 ml of 5% w / v silk solution and mix evenly; Step 3.2 Dissolve 5.7 g of Na3PO4 and 0.2 g of NaOH in 50 ml of deionized water and mix evenly; Step 3.3 Add dropwise the solution containing Na3PO4 and NaOH to the silk solution containing SrCl2 and CaCl2 to obtain a suspension, and adjust the pH to 11; Stir the suspension at 60°C for 24 hours for sufficient reaction, and age 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.

[0043] Step 4. Transmission electron microscopy (TEM) equipped with energy dispersive spectroscopy (EDS) was used to characterize the morphology and elemental composition of the Sr-SF-HA particles. X-ray diffraction (XRD) was used to evaluate the crystalline 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 materials were characterized by Fourier transform infrared spectroscopy (FTIR). Figure 2-Figure 5As shown. TEM detection revealed that the Sr-SF-HA particles were nanorod-like structures. In addition, the characteristic lattice spacing of hydroxyapatite was observed in the high-magnification image, including 0.334 nm corresponding to the (002) plane and 0.272 nm corresponding to the (211) plane. EDS detection revealed that the (Ca+Sr) / P ratio in Sr-SF-HA was 1.56, which was close to 1.67, which is almost the Ca / P ratio of natural hydroxyapatite. Full XPS spectroscopy revealed that the Sr-SF-HA nanoparticles contained the elements Sr, Ca, N, P, O, and C. Fine-spectral analysis revealed that the two main peaks of strontium, Sr3p and Sr3d, 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 C1s peaks can be fitted at 284.6 eV, 285.9 eV, and 287.8 eV, corresponding to the three main components, including C–C, C–O / C–N, and C=O / C=N bonds, respectively. X-ray diffraction (XRD) results showed that both SF-HA and Sr-SF-HA had characteristic diffraction peaks of hydroxyapatite crystals (JCPDS No. 09–0432) at (002), (210), (211), (300), (202), (310), (222), and (213), without any shifts or new peaks. These results indicate the successful synthesis of Sr-SF-HA nanoparticles.

[0044] Step 5. 3 g of 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. The uniform PCL / DCM solution was quickly poured into a glass dish and continuously stirred to evaporate the DCM to form a PCL composite film uniformly containing Sr-SF-HA nanoparticles.

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

[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 an 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 turns into 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 scaffold were characterized by field emission scanning electron microscopy equipped with energy dispersive spectroscopy (EDS). Figure 7 、 Figure 8 High-magnification surface and cross-sectional images reveal distinct Sr-SF-HA particles within the scaffold and a 1-2 μm thick nanostructured coating on the scaffold surface. EDS mapping images reveal that C, N, O, Ca, Sr, and P are uniformly distributed throughout the EP@PCL / Sr scaffold, demonstrating uniform EGCG / PEI coating and doping with Sr-SF-HA particles.

[0048] Step 9. Conduct experiments related to 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] In order to study the immunomodulatory effect of the scaffold, we first used flow cytometry to identify the phenotype of macrophages. First, macrophages were cultured at 1×10 5The cells were inoculated into 6-well plates at a density of 100 cells. Next, LPS (100 ng / mL) and IL-4 (20 ng / mL) were used to induce the cells for 48 hours, and then the original culture medium was removed. After further treatment with the culture medium containing each scaffold extract for 48 hours, the macrophages were resuspended in PBS. Then, the cells were incubated with the primary antibody in the dark for half an hour. Finally, the phenotype of the macrophages was analyzed by flow cytometry (CD86 corresponds to M1 type, CD206 corresponds to M2 type). 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 by immunofluorescence assay. Briefly, we seeded 2×10 4 Raw264.7 cells were collected and their original morphology was observed under a microscope, and images were collected. To test the regulatory effect of the material on M1 macrophages, all cells in the control group were treated with LPS solution at a concentration of 100 ng / mL for 48 hours. The treated cells were then observed and images were collected. The cells were washed with PBS and then cultured in culture medium containing different scaffold extracts for 48 hours. Cells without LPS served as the negative control group, and cells treated with LPS alone served as the positive control group. After treatment, the cells were fixed with 4% paraformaldehyde solution for 30 minutes, permeabilized with 0.5% TritonX-100, and incubated with 5% goat serum for 1.5 hours. They were then incubated with iNOS antibody (1:100) overnight. Next, the cells were incubated with secondary antibody (1:200) for 1 hour. Finally, DAPI stain was added and incubated in the dark for 15 minutes. iNOS expression was detected using a fluorescence microscope, and fluorescence intensity was semi-quantitatively analyzed using ImageJ. In order to detect the regulatory effect of the material on M2 macrophages, the cells of each group except the control group were treated with 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, and then the cells of each group were cultured for 48 hours using culture medium containing different scaffold extracts. The cells without IL-4 were used as the negative control group, and the cells with only IL-4 were used as the positive control group. After treatment, the cells were fixed with 4% paraformaldehyde solution for 30 minutes, penetrated with 0.5% Triton X-100, and incubated with 5% goat serum for 1.5 hours. Then incubated with iNOS antibody (1:100) overnight. Next, the cells were incubated with the second antibody (1:200) for 1 hour. Finally, DAPI stain was added and incubated in the dark for 15 minutes. The expression of CD206 was detected using a fluorescence microscope, and the fluorescence intensity was semi-quantitatively analyzed using ImageJ. The results are shown in Figure 2. Figure 9 As shown in Figure 2, the EP@PCL / Sr scaffold can effectively induce macrophage polarization to M2 and inhibit macrophage polarization to M1, demonstrating excellent immunomodulatory function.

[0051] To investigate the osteogenic differentiation-promoting effects of the scaffolds, rBMSCs were first seeded into 24-well plates and cultured to 80% confluence. Next, conventional culture medium was replaced with culture media containing various scaffold extracts. To accurately characterize the osteoinductive properties of the scaffolds, no external osteoinductive factors, such as β-glycerophosphate, vitamin C, and dexamethasone, were added. On day 7, the rBMSCs were fixed with 4% paraformaldehyde for 20 minutes and stained using the BCIP / NBT alkaline phosphatase assay. Furthermore, 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. The calcium nodules were then stained with Alizarin Red staining solution and observed using a stereomicroscope.

[0052] The expression of osteoblast-related proteins in rBMSCs was further studied by immunofluorescence. Briefly, polylysine-treated cell slides were placed in 12-well plates and rinsed with culture medium. After washing three times, the culture medium was aspirated for later use. Next, rBMSCs were seeded into the corresponding 12-well plates where cell slides were placed. Next, culture medium containing different scaffold extracts was used instead of conventional culture medium. On the 4th day, the cells were fixed with 4% paraformaldehyde for 30 minutes. Next, the cells were permeabilized with 0.5% TritonX-100 and incubated with 5% goat serum for 1.5 hours. They were then 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 cell nucleus was stained with DAPI. The expression of OPN and RUNX2 was detected using a fluorescence microscope, and the fluorescence intensity was semi-quantitatively analyzed using ImageJ. The results are shown in Figure 2. Figure 10 As shown; on the 7th day, the cells in the EP@PCL / Sr-treated group had darker blue staining than those in the CON group, indicating that the cells in the EP@PCL / Sr-treated group had stronger ALP activity and early osteogenic activity. On the 21st day, the cells in the EP@PCL / Sr-treated group produced a large number of calcium nodules, while only a small number of calcium nodules were produced in the CON group. This shows that the cells in the EP@PCL / Sr-treated group not only have stronger early osteogenic activity, but also lead to stronger osteogenic differentiation results. The expression levels of OPN and RUNX2 proteins in the cells of the EP@PCL / Sr-treated group were also significantly higher than those in the CON and PCL-treated groups. In summary, the EP@PCL / Sr scaffold has excellent ability to promote osteogenic differentiation.

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

[0054] For the tubule formation assay, the matrix gel was thawed overnight at 4°C. Next, the matrix gel was evenly added to a 24-well plate and incubated at 37°C for 30 minutes. Subsequently, cells were seeded into the corresponding wells and cultured in culture medium containing the 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 investigated by immunofluorescence. Briefly, poly-lysine-treated cell slides were placed in a 12-well plate and rinsed with culture medium. After washing three times, the culture medium was aspirated and set aside. 3 cells / well) were seeded into the corresponding 12-well plates with cell slides. Next, culture medium containing different scaffold extracts was used instead of regular culture medium. On the 4th day, the cells were fixed with 4% paraformaldehyde for 30 minutes. Next, the cells were permeabilized with 0.5% TritonX-100 and incubated with 5% goat serum for 1.5 hours. They were then incubated with VEGF antibody (1:100) overnight. 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 cell nuclei were stained with DAPI. The expression of VEGF was detected using a fluorescence microscope, and the fluorescence intensity was semi-quantitatively analyzed using ImageJ. The results are shown in Figure 2. Figure 11 The cells in the EP@PCL / Sr-treated group had stronger migration ability, the ability to promote tube formation, and significantly increased VEGF protein expression in endothelial cells, indicating that the scaffold has excellent angiogenic activity.

[0056] Application of the above-mentioned tea polyphenol coating-modified 3D-printed strontium-doped hydroxyapatite composite scaffold in restoring 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: Remove the sericin from the cocoon through a high-temperature alkaline degumming process to obtain silk fibroin for use; Step 2: dissolving the silk fibroin obtained in step 1 in a predetermined amount of lithium bromide aqueous solution to obtain a liquid, transferring the liquid into a dialysis bag, and dialyzing the mixture against deionized water for 3 days to remove the lithium bromide; The silk fibroin solution was obtained and stored at 4°C until use; Step 3. Synthesize Sr-SF-HA nanoparticles by hydrothermal coprecipitation using CaCl2, SrCl2, Na3PO4 and the silk fibroin solution from step 2; Step 4. Characterize the morphology and elemental composition of the Sr-SF-HA nanoparticles obtained in Step 3 using a transmission electron microscope equipped with energy dispersive spectroscopy (EDS); Step 5. Disperse 3-6 g of Sr-SF-HA nanoparticles obtained in Step 3 evenly in 40-60 ml of dichloromethane. Then, add 10-20 g of PCL particles. After the PCL particles are completely dissolved, a uniform solution is prepared. The uniform PCL / DCM solution is quickly poured into a glass dish and continuously stirred to evaporate the DCM, thereby forming a PCL composite film uniformly containing the Sr-SF-HA nanoparticles. Step 6. Use software to design the parameters of the porous cylindrical scaffold on a computer. Then, load the PCL composite membrane containing Sr-SF-HA nanoparticles from Step 5 into a high-temperature barrel and heat it to 120°C. Simultaneously, set the printing parameters and, after the raw material is completely melted, proceed with printing to obtain the 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. The PCL / Sr scaffold was immersed in the above EGCG / PEI coating solution for 4 hours. Then, the PCL / Sr scaffold was washed with deionized water and dried to obtain the EP@PCL / Sr scaffold with EGCG / PEI coating; the pH value of Tris buffer was 8.5; Step 8. Characterize the morphology and elemental composition of the EP@PCL / Sr scaffolds using a field emission scanning electron microscope equipped with an 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: placing the cocoons in a 2% w / v sodium carbonate solution at 120° C. for 30 minutes, then washing them three times with deionized water to remove the sericin and leave the silk fibroin, and then drying the silk fibroin in an oven for later use.

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

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