Preparation method and application of a bionic scaffold with bone repair and anti-osteosarcoma functions

Abstract

The application relates to a preparation method and application of a bionic scaffold with bone repair and anti-osteosarcoma functions, and the specific steps are as follows: firstly, polydopamine nanoparticles are added into a curcumin ethanol solution, room temperature stirring reaction is carried out, and a polydopamine nanoparticle solution loaded with curcumin is obtained; secondly, freeze-dried silk fibroin is dissolved in hexafluoroisopropanol to form a silk fibroin solution, acetone is added to crosslink the solution into a gel, ammonium bicarbonate is added, hydroxyapatite nanoparticles are added, and mixing is uniformly carried out, and a porous fiber scaffold is synthesized through supercritical CO2 assisted phase separation; finally, the porous fiber scaffold is added into the polydopamine nanoparticle solution loaded with curcumin, room temperature stirring reaction is carried out, and then freeze-drying is carried out, so that the silk fibroin scaffold is obtained. The silk fibroin scaffold provided by the application has the anti-osteosarcoma function, can simulate the composition and structure of bone and induce new bone formation, can simultaneously meet the purposes of treating bone tumors and repairing bone defects, and has important clinical value and scientific significance.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a method for preparing and applying a biomimetic scaffold that combines bone repair and anti-osteosarcoma properties. Background Technology

[0002] Bone tumors generally refer to malignant tumors that occur in the bone or its surrounding tissues. For example, osteosarcoma is a highly metastatic malignant tumor with a 5-year survival rate of less than 20%. Currently, the main treatment for bone tumors is a combination of surgery, radiation, and chemotherapy. When bone tumor surgery causes large bone defects, bone grafts are needed to repair them. Autologous bone grafting is considered the "gold standard" bone graft material, but its sources are limited and it can cause secondary surgical trauma. Allogeneic grafts have a wider range of sources, but there is a possibility of immune rejection and disease transmission.

[0003] Natural bone is primarily an organic / inorganic complex composed of nano-hydroxyapatite (nHA) and collagen fibers (50–500 nm in diameter). Synthetically synthesized nHA is similar in composition and structure to the bone matrix, exhibiting good biocompatibility and osteoconductivity. It can tightly bind to bone formation and promote bone growth, making it a widely used bone repair material. Silk fibroin (SF) has been extensively studied as a bone repair material. SF possesses good biocompatibility and degradability, and does not cause coagulation, immune, or inflammatory reactions. In particular, SF's structure is similar to bone collagen without significant antigenicity, and it has better toughness than commonly used collagen. Inorganic components such as calcium phosphate can deposit on SF, and the two have a strong binding force. Furthermore, SF possesses excellent mechanical properties, can promote angiogenesis, induce the adhesion and growth of bone mesenchymal stem cells (BMSCs), and has a certain degree of osteoinductive properties. Therefore, constructing silk fibroin / nanohydroxyapatite (SF / nHA) composite scaffold materials can effectively mimic the composition of natural bone. For example, the patent document with publication number CN110180029A uses silk fibroin solution and nanohydroxyapatite as the main raw materials to prepare a biodegradable material with osteogenic differentiation and bone regeneration functions.

[0004] For bone defects caused by bone tumor surgery, existing bone repair scaffolds can fill and induce repair, but they are ineffective against any residual bone tumor cells. Therefore, there is a clinical need to construct tissue-engineered scaffold materials that mimic the composition and structure of bone to meet this requirement. Summary of the Invention

[0005] This invention provides a method for preparing and applying a biomimetic scaffold that combines bone repair and anti-osteosarcoma properties. Supercritical CO2 technology allows for precise control of the porosity and porous morphology of the silk fibroin scaffold, forming a solvent-free scaffold under reduced pressure. This method produces a scaffold that closely resembles the structure and composition of bone, and the scaffold contains curcumin-adsorbed polydopamine nanoparticles, exhibiting anti-bone tumor capabilities. Furthermore, it mimics the composition and structure of bone and induces new bone formation, simultaneously achieving the goals of treating bone tumors and repairing bone defects, thus possessing significant clinical value and scientific significance.

[0006] The present invention provides the following solution to the above-mentioned technical problems: a method for preparing a biomimetic scaffold that combines bone repair and anti-osteosarcoma properties, comprising the following steps:

[0007] 1) Add polydopamine nanoparticles to a curcumin ethanol solution and stir at room temperature to obtain a curcumin-loaded polydopamine nanoparticle solution. The specific operation is as follows:

[0008] Weigh 60 mg of dopamine hydrochloride and add it to 120 ml of deionized water. After stirring, add 1.2 mL of Tris-HCl buffer (100×) to adjust the pH to 8.5 to initiate the reaction. After reacting at room temperature for 24 h, transfer the reaction liquid to a centrifuge tube and separate the product from the unreacted liquid using a high-speed centrifuge. Wash the product three times with deionized water and anhydrous ethanol, and centrifuge three times to obtain polydopamine nanoparticles. Weigh 40 mg of curcumin and dissolve it in 40 ml of anhydrous ethanol. After stirring, add the previously obtained polydopamine nanoparticles to the curcumin solution and react in the dark for 12 h. Transfer the reaction liquid to a centrifuge tube and separate the product from the unreacted liquid using a high-speed centrifuge. Wash the product three times with deionized water and centrifuge three times to obtain a curcumin-loaded polydopamine nanoparticle solution. Freeze-dry the solution to obtain dried CM-PDA nanoparticles.

[0009] 2) The freeze-dried silk fibroin was dissolved in hexafluoroisopropanol to form a silk fibroin solution. Acetone was added to crosslink and form a gel. Ammonium bicarbonate was added, and then nano-hydroxyapatite was added. The mixture was mixed evenly and a porous fiber scaffold was synthesized by supercritical CO2-assisted phase separation.

[0010] The freeze-dried silk fibroin preparation process is as follows: Approximately 10g of raw silk is weighed and placed in a 0.02M sodium carbonate solution. The solution is heated in a water bath at 100℃ for 2 hours to remove the outer layer of sericin from the raw silk. The degummed silk fibroin fibers are washed three times with deionized water, and the above steps are repeated twice. Then, the dried silk fibroin fibers are dissolved in a ternary CaCl2 / H2O / ethanol solution and heated in a water bath to 80℃ for 3 hours to obtain a silk fibroin solution. Finally, the silk fibroin solution is dialyzed against deionized water for 5 days to remove impurity ions. The water is changed every 4 hours for the first three days, and then three times a day thereafter. The dried silk fibroin can then be further obtained through freeze-drying.

[0011] The synthesis of porous fiber scaffolds by supercritical CO2-assisted phase separation involves mixing the above-mentioned materials evenly and placing them into a polytetrafluoroethylene mold. Then, the mold is placed into the reactor of a supercritical device (such as the SFE-2 model supercritical carbon dioxide equipment), carbon dioxide is introduced, and the reaction temperature, pressure, and time are set to obtain the porous fiber scaffold.

[0012] 3) The porous fiber scaffold was added to a solution of curcumin-loaded polydopamine nanoparticles, stirred at room temperature for 12 hours, and then freeze-dried to obtain the silk fibroin scaffold.

[0013] Preferably, in step 1), the concentration of the curcumin ethanol solution is 0.8-1.2 mg / mL; more preferably, in step 1), the concentration of the curcumin ethanol solution is 1 mg / mL.

[0014] In step 1), each 1 mg of polydopamine nanoparticles is loaded with 0.75-1 mg of curcumin.

[0015] Preferably, the reaction time in step 1) is 12-24 hours; more preferably, the reaction time in step 1) is 24 hours.

[0016] Preferably, in step 2), the concentration of the silk fibroin solution is 8-12 g / mL; more preferably, in step 2), the concentration of the silk fibroin solution is 10 g / mL.

[0017] Preferably, in step 2), 0.24-0.28 mL of acetone is added to every 1 mL of silk fibroin solution.

[0018] Preferably, in step 2), 0.8-1.2g of ammonium bicarbonate is added for every 1g of freeze-dried silk fibroin; the diameter of the ammonium bicarbonate is 150-250μm; preferably, in step 2), 1g of ammonium bicarbonate is added for every 1g of freeze-dried silk fibroin; the diameter of the ammonium bicarbonate is 150-250μm.

[0019] Preferably, in step 2), the amount of nano-hydroxyapatite added is 1-5 wt%, based on the amount of freeze-dried silk fibroin protein; more preferably, in step 2), the amount of nano-hydroxyapatite added is 1 wt%, based on the amount of freeze-dried silk fibroin protein.

[0020] Preferably, in step 2), the reaction temperature for supercritical CO2-assisted phase separation is set to 35°C, the reaction pressure to 15 MPa, and the reaction time to 3-6 h.

[0021] The application of silk fibroin scaffolds prepared by the supercritical CO2 technology method described above in bone graft materials.

[0022] The beneficial effects of this invention are as follows:

[0023] 1. This invention utilizes a supercritical CO2-assisted phase separation method, introducing ammonium bicarbonate as a pore-forming agent to precisely control the porosity and porous morphology of the scaffold, and forming a novel solvent-free SF / nHA bioactive biomimetic scaffold under reduced pressure. It not only mimics the composition of bone but also structurally imitates the collagen nanofiber network in the natural extracellular matrix, achieving a bone-like nano / micro-level porous structure.

[0024] 2. The SF / nHA bioactive biomimetic scaffold prepared in this invention adsorbs curcumin-loaded polydopamine nanoparticles. Curcumin (CM) possesses pharmacological effects such as antitumor, anti-inflammatory, anticoagulant, and lipid-lowering properties. Curcumin can inhibit the growth of various cancers, including osteosarcoma, liver cancer, gastric cancer, lung cancer, colon cancer, and multiple myeloma, with minimal toxic side effects on the human body and is inexpensive. By loading curcumin onto polydopamine (PDA), its stability can be maintained, further enhancing its highly effective and low-toxicity antitumor properties. Polydopamine nanoparticles exhibit strong near-infrared absorption, high photothermal conversion efficiency, and significant biocompatibility and biodegradability. Combined with photothermal-chemotherapy, it can further eliminate residual osteosarcoma tissue after surgery. The photothermal effect can ablate osteosarcoma cells through the generated high temperature, while also increasing the penetration rate and release rate of curcumin, effectively treating osteosarcoma.

[0025] In summary, the silk fibroin scaffold prepared by the supercritical CO2-assisted phase separation method of this invention can not only mimic the composition of bone, but also structurally mimic the collagen nanofiber network in the natural extracellular matrix, and obtain a bone-like nano / micro-level porous structure. The scaffold has excellent photothermal properties, and can effectively treat osteosarcoma through photothermal-chemotherapy synergy. Through its excellent photothermal performance, the scaffold can provide effective antibacterial effects, and as an implantable scaffold, it can effectively avoid infection at the implantation site, while also enabling the scaffold to better treat osteosarcoma and promote bone repair.

[0026] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Specific embodiments of the present invention are given in detail below with reference to the accompanying drawings. Attached Figure Description

[0027] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings:

[0028] Figure 1 Scanning electron microscope image of the CM-PDA / SF / 1%nHA scaffold prepared in Example 2;

[0029] Figure 2 The porosity histograms are for the silk fibroin scaffolds prepared in Examples 1-4.

[0030] Figure 3 Bar charts showing the compressive strength of the silk fibroin scaffolds prepared in Examples 1-4;

[0031] Figure 4 The CM-PDA / SF / 1%nHA stent prepared for Example 2 was tested at different near-infrared powers (0.5, 0.75, 1.0, and 1.5 W cm⁻¹). -2 A line graph showing the temperature change under irradiation;

[0032] Figure 5 The image shows a bar chart of bone volume after scaffold implantation in the skull of rats in Example 5.

[0033] Figure 6 This is a bar chart showing the tumor volume of each group of mice after scaffold implantation in Example 6;

[0034] Figure 7 This is a bar chart showing the body weight of mice after scaffold implantation into mouse tumors in each group in Example 6;

[0035] Figure 8 These are colony photographs of each group of scaffolds after culturing with Escherichia coli and Staphylococcus aureus in Example 7. Detailed Implementation

[0036] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0037] Example 1

[0038] This embodiment provides a CM-PDA / SF stent prepared based on supercritical CO2 technology, the preparation method of which includes the following steps:

[0039] 1) Weigh 60 mg of dopamine hydrochloride and add it to 120 ml of deionized water. After stirring well, add 1.2 mL of Tris-HCl buffer (100×) to adjust the pH to 8.5 to initiate the reaction. After reacting at room temperature for 24 h, transfer the reaction liquid to a centrifuge tube and separate the product from the unreacted liquid using a high-speed centrifuge. Wash the product three times with deionized water and anhydrous ethanol, and centrifuge three times to obtain polydopamine nanoparticles.

[0040] Weigh 40 mg of curcumin and dissolve it in 40 ml of anhydrous ethanol. After stirring evenly, add the previously obtained polydopamine nanoparticles to the curcumin solution and react in the dark for 12 hours. Transfer the reaction liquid to a centrifuge tube and separate the product from the unreacted liquid using a high-speed centrifuge. Wash the product with deionized water three times and centrifuge each time. After freeze-drying, a dry curcumin-loaded polydopamine nanoparticle solution is obtained, denoted as CM-PDA nanoparticle solution.

[0041] 2) Weigh approximately 10g of raw silk and place it in a 0.02M sodium carbonate solution. Heat the solution in a water bath at 100℃ for 2 hours to remove the sericin from the outer layer of the raw silk. Wash the degummed silk fibroin fibers three times with deionized water, and repeat the above steps twice. Then, dissolve the dried silk fibroin fibers in a ternary CaCl2 / H2O / ethanol solution and heat it in a water bath to 80℃ for 3 hours to obtain a silk fibroin solution. Finally, dialyze the silk fibroin solution with deionized water for 5 days to remove impurity ions. Change the water every 4 hours for the first three days, and then change the water 3 times a day thereafter. Freeze-drying can be used to further obtain freeze-dried silk fibroin.

[0042] Lyophilized silk fibroin was dissolved in hexafluoroisopropanol to form a silk fibroin solution with a concentration of 10 g / ml. Acetone was then added at a ratio of 1.2-1.4 ml per 5 mL of silk fibroin solution to crosslink and form a gel. Ammonium bicarbonate with a diameter of 150-250 micrometers was added as a pore-forming agent at a mass ratio of 1:1 to the silk fibroin solution, and the mixture was rapidly and thoroughly stirred until homogeneous. The mixture was placed in a polytetrafluoroethylene mold and then placed in a supercritical fluid reactor. Carbon dioxide was introduced, and the parameters were adjusted to 35℃ and 15 MPa for a reaction time of 3-6 hours to obtain a silk fibroin scaffold, denoted as the SF scaffold.

[0043] 3) Add the SF scaffold obtained in step 2) to the CM-PDA nanoparticle solution obtained in step 1), stir at room temperature for 12 h, and freeze dry to obtain a silk fibroin composite scaffold loaded with curcumin-modified polydopamine nanoparticles, denoted as CM-PDA / SF scaffold.

[0044] Example 2

[0045] This embodiment provides a CM-PDA / SF / 1%nHA scaffold prepared based on supercritical CO2 technology. The preparation method includes the following steps, which are basically the same as in Example 1, except that in step 2), after adding ammonium bicarbonate, 1 wt% of nano-hydroxyapatite is added based on the mass of the silk fibroin solution, and the mixture is rapidly and thoroughly stirred until homogeneous. The above mixture is placed in a polytetrafluoroethylene mold, and then placed in the reactor of a supercritical device. Carbon dioxide is introduced, and the parameters are adjusted to 35℃ and 15 MPa. The reaction time is 3–6 h, thus obtaining a silk fibroin composite scaffold doped with nano-hydroxyapatite and loaded with curcumin-modified polydopamine nanoparticles, denoted as the CM-PDA / SF / 1%nHA scaffold. The CM-PDA / SF / 1%nHA scaffold is imaged using a scanning electron microscope, as shown below. Figure 1 As shown, the nanofiber structure of the CM-PDA / SF / 1%nHA scaffold is beneficial for cell seeding and penetration.

[0046] Example 3

[0047] This embodiment provides a CM-PDA / SF / 3%nHA scaffold prepared based on supercritical CO2 technology. The preparation method includes the following steps, which are basically the same as those in Example 2, except that the amount of nano-hydroxyapatite is 3wt%.

[0048] Example 4

[0049] This embodiment provides a CM-PDA / SF / 5%nHA scaffold prepared based on supercritical CO2 technology. The preparation method includes the following steps, which are basically the same as those in Example 2, except that the amount of nano-hydroxyapatite is 5wt%.

[0050] The stents were immersed in a known volume (V1) of deionized water for 30 minutes until they were saturated. The total volume of deionized water containing the stents was recorded as (V2). Then, after the stents were removed, the remaining volume of deionized water was recorded as (V3). Finally, the porosity of the stents was calculated using the following equation:

[0051] Porosity = (V1-V3) / (V2-V3)*100%

[0052] The porosity of the stents obtained in Examples 1-4 was measured, such as... Figure 2 As shown, the percentages were 63.0%, 68.8%, 69.6%, and 77.3%, respectively.

[0053] The support was fabricated into a cylinder with a diameter of 10 mm and a height of 10 mm using an electronic universal testing machine. A 0.1 kN sensor was used in the experiment, and the support was compressed at a speed of 1 mm / min. The compressive strength of the support was determined according to ISO 604:2002, with 25% compressive strain defined as its compressive strength. Each group was repeated five times, and the compressive strength of the supports obtained in Examples 1-4 was measured. Figure 3 As shown, the values ​​are 0.69, 1.41, 0.88, and 0.32 MPa, respectively.

[0054] In Example 2, the doping amount of nano-hydroxyapatite significantly improved the mechanical properties of the scaffold. As the doping amount of nano-hydroxyapatite continued to increase, the compressive strength of the scaffold gradually decreased. Therefore, the scaffold of Example 2 exhibited significantly better mechanical properties and possessed suitable porosity, thus better meeting the requirements for bone regeneration.

[0055] The scaffold prepared in Example 2 (cut to a diameter of 5 mm) was placed in 200 μl of deionized water and subjected to power levels of 0.5, 0.75, 1.0, and 1.5 W / cm², respectively. 2 The deionized water was irradiated with a near-infrared laser for 600 seconds, and the temperature was recorded every 60 seconds. Figure 4 As shown, the water temperature varies with different power densities, exhibiting power density-dependent photothermal properties. Furthermore, the scaffold temperature rapidly rises to over 50°C, and this stable temperature can directly lead to cell apoptosis or necrosis.

[0056] Example 5

[0057] Twenty-four female and twenty-four male SD rats (4 weeks old, 200-220g) were randomly divided into four groups. The rats were anesthetized intraperitoneally with sodium phenobarbital (100mg / kg). A circular drill was then used to create a circular defect (d=5mm) in the sagittal suture of the skull on one side. During drilling, the drilling site was continuously flushed with physiological saline to remove blood and bone fragments. Simultaneously, the drilling process was kept cool to prevent excessive heat from causing further harm to the rats.

[0058] The treatments for each group were as follows: 1. No blank (control group);

[0059] 2. Fill with SF stent (using the SF stent prepared in step 2 of Example 1);

[0060] 3. Fill the CM-PDA / SF stent (the stent prepared in Example 1);

[0061] 4. Filling with CM-PDA / SF / 1% nHA scaffolds (scaffolds prepared in Example 2). Each scaffold was 5 mm in diameter and 1 mm thick. Four and eight weeks after implantation, a batch of mice were sacrificed, and the skulls were removed and preserved in 4% neutral formalin buffer for 2 days. The skull defects with scaffolds were decalcified in 10% EDTA / HCl for 30 days, dehydrated by gradient ethanol, and embedded in paraffin. The coverage area of ​​regenerated bone tissue was measured 8 weeks after implantation using ImageJ software. Figure 5 As shown, the results indicate that the bone repair scaffold obtained in this invention can effectively promote bone tissue regeneration, and the bone regeneration tissue of the CM-PDA / SF / 1%nHA scaffold implanted in Example 2 is the best.

[0062] Example 6

[0063] Thirty 6-week-old Balb / c female mice weighing 18-20g were randomly divided into five groups: control group, SF group (SF scaffold implanted at the tumor site), CM-PDA / SF / 1%nHA scaffold group (CM-PDA / SF / 1%nHA scaffold implanted at the tumor site), PDA / SF / 1%nHA+NIR group (PDA / SF / 1%nHA scaffold implanted at the tumor site and subjected to 10 minutes of NIR irradiation; the preparation method of PDA / SF / 1%nHA scaffold was basically the same as that of CM-PDA / SF / 1%nHA scaffold, except that the polydopamine nanoparticles prepared in step 1 were not loaded with curcumin, and the PDA nanoparticle solution was mixed with SF / 1%nHA in step 3 and then freeze-dried to prepare the PDA / SF / 1%nHA scaffold), and CM-PDA / SF / 1%nHA+NIR group (CM-PDA / SF / 1%nHA scaffold implanted at the tumor site and subjected to 10 minutes of NIR irradiation).

[0064] After digestion and centrifugation, the cultured K7M2 WT cells were resuspended in PBS buffer to a concentration of 4 × 10⁻⁶. 6 100 μl / mouse. The backs of Balb / c mice were shaved, and 100 μl of the prepared K7M2 WT cell suspension was injected into the backs of each mouse. The mice were observed daily for tumor growth and tumor volume was measured. Tumors were counted until they reached 120 mm². 3Around 10:00 AM, the tumor-bearing site was incised and a scaffold was implanted in the mice. Balb / c mice were anesthetized via intraperitoneal injection of 2% sodium pentobarbital (30 mg / ml). The hair around the tumor was shaved, and a scalpel was used to make an incision at the tumor site, exposing only the subcutaneous tissue. The scaffold was fixed in situ to the tumor. Mice requiring NIR irradiation were irradiated with 808 nm near-infrared light for 10 minutes, and the incision was sutured. After scaffold implantation, the weight of each mouse was measured and the tumor size was calculated every two days. Seven days later, three mice from each group were randomly sacrificed using an overdose of anesthesia, and the tumor tissue was removed, photographed, and stained with H&E. Fourteen days later, all remaining mice were sacrificed using an overdose of anesthesia, and the tumor tissue was removed, photographed, and stained with H&E. Tumor volume changes are shown below. Figure 6 As shown, the results indicate that tumor volume decreased in the PDA / SF / 1%nHA+NIR group and the CM-PDA / SF / 1%nHA+NIR group, demonstrating that the scaffold obtained in this invention combined with photothermal-chemotherapy can effectively treat osteosarcoma. The body weight of each group of mice was recorded every two days, as shown below. Figure 7 As shown, the weight of each group of mice did not decrease significantly, indicating that the scaffold obtained in this invention has no obvious toxicity to mice and has good biocompatibility.

[0065] Example 7

[0066] In 48-well cell culture plates, sterilized with ultraviolet light for 24 hours, the cells were divided into four experimental groups: 1) 20 μL of diluted bacterial suspension (10) was added to a scaffold-free 48-well cell culture plate. 8 1) Add 20 μL of bacterial suspension to a scaffold-free 48 cell culture plate and use a near-infrared laser (808 nm, 0.75 W / cm²) to infuse the solution. 2 1) Irradiate for 10 min; 2) Add 20 μL of diluted bacterial suspension to a sterile CM-PDA / SF / 1% nHA scaffold; 3) Add 20 μL of diluted bacterial suspension (10 8 CFU / mL was added to a sterile CM-PDA / SF / 1% nHA scaffold and treated with a near-infrared laser (808nm, 0.75W / cm²). 2 Irradiate for 10 min. Then, incubate for 2 h in a shaking incubator at 100 rpm / min and 37°C. Wash each group of materials with 1 ml of sterile PBS; then evenly disperse 100 μL of bacterial suspension on agar plates and incubate for 12 h in a constant temperature shaking incubator. The bacterial suspensions are divided into two types: Escherichia coli suspension and Staphylococcus aureus suspension.

[0067] Finally, the antimicrobial properties of the material were analyzed by photographing and counting the colonies on the agar plates. Colony counts on the agar plates are shown below. Figure 8The results show that the stent obtained by the present invention has excellent antibacterial properties under near-infrared laser conditions.

[0068] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Those skilled in the art can readily implement the present invention based on the accompanying drawings and the above description. However, any modifications, alterations, or variations made by those skilled in the art without departing from the scope of the present invention, utilizing the disclosed technical content, are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, or variations made to the above embodiments based on the essential technology of the present invention are still within the protection scope of the present invention.

Claims

1. A method for preparing a biomimetic scaffold that combines bone repair and anti-osteosarcoma properties, characterized in that, Includes the following steps: 1) Polydopamine nanoparticles were added to a curcumin ethanol solution and stirred at room temperature to obtain a curcumin-loaded polydopamine nanoparticle solution. The specific operation is as follows: The preparation process of polydopamine nanoparticles is as follows: 60 mg of dopamine hydrochloride was weighed and added to 120 ml of deionized water. After stirring evenly, 1.2 mL of 100×Tris-HCl buffer was added to adjust the pH to 8.5 to initiate the reaction. After reacting at room temperature for 24 h, the reaction liquid was transferred to a centrifuge tube, and the product was separated from the unreacted liquid using a high-speed centrifuge. The product was washed three times with deionized water and anhydrous ethanol respectively and centrifuged to obtain polydopamine nanoparticles. 2) The freeze-dried silk fibroin was dissolved in hexafluoroisopropanol to form a silk fibroin solution. Acetone was added to crosslink and form a gel. Ammonium bicarbonate was added. Based on the amount of freeze-dried silk fibroin, 1-5 wt% of nano-hydroxyapatite was added and mixed evenly. Porous fiber scaffolds were synthesized by supercritical CO2-assisted phase separation. The reaction temperature was set at 35 ℃, the reaction pressure at 15 MPa, and the reaction time at 3-6 h. The preparation process of freeze-dried silk fibroin is as follows: Weigh about 10 g of raw silk and put it into a 0.02 M sodium carbonate solution. Heat the solution in a water bath at 100°C for 2 hours to remove the sericin from the outer layer of the raw silk. Wash the degummed silk fibroin fibers three times with deionized water. Repeat the above steps twice. Then, dissolve the dried silk fibroin fibers in a ternary system of CaCl2 / H2O / ethanol solution and heat it in a water bath to 80°C for 3 hours to obtain a silk fibroin solution. Finally, dialyze the silk fibroin solution with deionized water for 5 days to remove impurity ions. Change the water every 4 hours for the first three days and then change the water 3 times a day thereafter. Further obtain dried silk fibroin by freeze-drying. 3) The porous fiber scaffold was added to a solution of polydopamine nanoparticles loaded with curcumin, stirred at room temperature and then freeze-dried to obtain the biomimetic scaffold.

2. The method for preparing a biomimetic scaffold combining bone repair and anti-osteosarcoma properties according to claim 1, characterized in that, In step 1), the concentration of the curcumin ethanol solution is 0.8-1.2 mg / mL.

3. The method for preparing a biomimetic scaffold combining bone repair and anti-osteosarcoma properties according to claim 1, characterized in that, In step 1), each 1 mg of polydopamine nanoparticles is loaded with 0.75-1 mg of curcumin.

4. The method for preparing a biomimetic scaffold combining bone repair and anti-osteosarcoma properties according to claim 1, characterized in that, Step 1), the reaction time is 12-24 h.

5. The method for preparing a biomimetic scaffold combining bone repair and anti-osteosarcoma properties according to claim 1, characterized in that, In step 2), add 0.24-0.28 mL of acetone to every 1 mL of silk fibroin solution.

6. The method for preparing a biomimetic scaffold combining bone repair and anti-osteosarcoma properties according to claim 1, characterized in that, In step 2), 0.8-1.2 g of ammonium bicarbonate is added for every 1 g of freeze-dried silk fibroin; the diameter of the ammonium bicarbonate is 150-250 μm.

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