Three-dimensional stent, preparation method therefor, and use thereof

Abstract

The present application discloses a three-dimensional stent, a preparation method therefor, and use thereof. Starting materials for preparing the three-dimensional stent provided by the present application include a carbon-inorganic oxide hybrid micro-nanofiber and carrageenan. The three-dimensional stent provided by the present application not only enables photo-thermal treatment, photo-thermal antibacterial effects, controlled drug release, and bone repair, but also has excellent biocompatibility and biological activity.

Description

Three-dimensional scaffold and preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, in particular to a three-dimensional scaffold and a preparation method and application thereof. BACKGROUND

[0002] Bone tumor refers to a tumor occurring in bone or originating from bone tissue components (bone and accessory tissues). The incidence of primary bone tumor in China is about 2-3 cases per 100,000 population, and about 40% of them are malignant tumors. At present, the treatment of bone tumor patients mainly relies on surgical resection of tumors, supplemented by multidisciplinary comprehensive treatment methods including immunotherapy, gene therapy and targeted therapy. Targeted therapy is a method of increasing the local concentration of drugs in the target area by combining specific carriers with drugs, so as to improve the therapeutic effect. However, surgical resection, radiotherapy and other treatment methods have the limitations of large trauma, difficulty in completely removing diseased tissues and easy recurrence. The postoperative oral or injection drug treatment method has the following problems: first, it is difficult to maintain an effective drug concentration in the lesion area; second, long-term use of this treatment method may cause toxic side effects, resulting in unsatisfactory treatment effect.

[0003] Therefore, it is necessary to provide a three-dimensional scaffold with controllable drug release and bone repair and photothermal therapy.

[0004] SUMMARY

[0005] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the first aspect of the present application provides a three-dimensional scaffold having the effects of photothermal therapy, photothermal antibiosis, drug controlled release and bone repair.

[0006] The second aspect of the present application further provides a preparation method of the three-dimensional scaffold.

[0007] The third aspect of the present application further provides an application of the three-dimensional scaffold.

[0008] The three-dimensional scaffold provided by the first aspect of the present application has the effects of photothermal therapy, photothermal antibiosis, drug controlled release and bone repair.

[0009] The three-dimensional scaffold according to the embodiments of the present application has at least the following beneficial effects:

[0010] The three-dimensional scaffold provided by the present application not only has the effects of photothermal therapy, photothermal antibiosis, drug controlled release and bone repair, but also has excellent biocompatibility and biological activity.

[0011] Further, the introduction of carbon hybrid energy improves the photothermal therapy, photothermal sterilization and drug loading capacity of inorganic micro-nano fibers, and the presence of bioactive inorganic oxides makes the carbon-inorganic oxide hybrid micro-nano fibers have good bioactivity and bioactivity. The three-dimensional scaffold of the present application has sufficient porosity to facilitate cell material exchange, which is beneficial to cell growth, and the microporous structure of carbon also has a good effect on adsorbing drugs; has stable mechanical properties, which has a certain supporting effect on cell growth and adsorbed drugs; has good biocompatibility and bioactivity, and has excellent photothermal performance, so it can be used for photothermal antibacterial and photothermal tumor inhibition; the drug release system with carrageenan as the matrix can adjust the drug release rate by changing the temperature, and when the scaffold receives infrared light irradiation, the temperature of the carrageenan in the scaffold can be changed, thereby controlling the release of drugs by controlling the swelling of the carrageenan, effectively avoiding the occurrence of excessive drugs or low drug concentration, thereby improving the treatment effect.

[0012] According to some embodiments of the present application, the three-dimensional scaffold has a macroporous structure of 30-500 pm, a fiber structure of 200 nm-3000 nm, and a porosity of 70%-98%.

[0013] According to some embodiments of the present application, the carbon-inorganic oxide hybrid micro-nano fiber is prepared by the following method:

[0014] S1, mixing and stirring a silicon source, a phosphorus source, ethanol, a calcium source, an acid catalyst and water to obtain a precursor solution, mixing a template polymer solution and the precursor solution to obtain a spinning solution;

[0015] S2, electrospinning the spinning solution, and then carbonizing to obtain a carbon-inorganic oxide hybrid micro-nano fiber.

[0016] According to some embodiments of the present application, the mass ratio of the template polymer solution to the precursor solution is 1:10-1:0.1.

[0017] According to some embodiments of the present application, in step S2, the parameters of electrospinning are set as follows:

[0018] The spinning voltage is 10-30 kV, the receiving distance is 5-30 cm, the perfusion speed is 0.5-5 mL / h, the temperature is 15-35℃, and the relative humidity is 25%-60%.

[0019] According to some embodiments of the present application, in step S2, pre-oxidation is performed before carbonization.

[0020] According to some embodiments of the present application, in step S2, the pre-oxidation temperature is 150-400℃.

[0021] According to some embodiments of the present application, in step S2, the pre-oxidation time is 0.5-4h.

[0022] According to some embodiments of the present application, in step S2, the carbonization temperature is 400-800℃.

[0023] According to some embodiments of the present application, in step S2, the carbonization time is 1-8h.

[0024] According to some embodiments of the present application, the calcium source is at least one of calcium gluconate, calcium chloride, calcium nitrate tetrahydrate, calcium bromide, calcium iodide, calcium nitrate, calcium dihydrogen phosphate, calcium bicarbonate, calcium bisulfite, calcium bisulfite, calcium hypochlorite, calcium chlorate, calcium perchlorate, and calcium permanganate.

[0025] According to some embodiments of the present application, the silicon source is at least one of tetraethyl orthosilicate, tetrapropyl orthosilicate, tetrabutyl orthosilicate, and polyethyl silicate.

[0026] According to some embodiments of the present application, the phosphorus source is at least one of triethyl phosphate and n-butyl phosphate.

[0027] According to some embodiments of the present application, the acid catalyst is at least one of formic acid, acetic acid, hydrochloric acid, nitric acid, phosphoric acid, polyphosphoric acid, oxalic acid, propionic acid, butyric acid, octanoic acid, adipic acid, oxalic acid, malonic acid, succinic acid, and maleic acid.

[0028] According to some embodiments of the present application, the template polymer in the template polymer solution includes at least one of cellulose acetate, ethyl cellulose, hydroxypropyl cellulose, chitin, chitosan, polyvinyl alcohol, polyvinyl butyral, polyacrylic acid, gelatin, polyethylene oxide, polyvinylidene fluoride, polyvinylpyrrolidone, polylactic acid, polycaprolactone, polyglycolide, polyurethane, polyacrylonitrile, polyvinyl acetate, polyacrylamide, polycarbonate, polyimide, polyetherimide, polyvinylidene fluoride, and polycyclooxanone.

[0029] According to some embodiments of the present application, the concentration of the template polymer solution is 2wt%-20wt%.

[0030] According to some embodiments of the second aspect of the present application, a method for preparing the three-dimensional scaffold is provided, including the following steps:

[0031] (1) mixing the carbon-inorganic oxide hybrid micro-nano fiber, carrageenan, and water, and uniformly dispersing to obtain a mixture;

[0032] (2) freezing the mixture, and then freeze-drying to obtain the three-dimensional scaffold.

[0033] According to some embodiments of the present application, the mass ratio of the carbon-inorganic oxide hybrid micro-nano fiber and the carrageenan is 1: (0.1-10).

[0034] The third aspect of the present application provides a use of the three-dimensional scaffold as described above in the preparation of a medical material for treating osteosarcoma.

[0035] Other features and advantages of the present application will be set forth in the following description, and in part will become apparent from the description, or can be learned by practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0036] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the description of the embodiments, taken in conjunction with the following drawings in which:

[0037] FIG. 1A is a photograph of the carbon-inorganic oxide hybrid micro-nano fiber prepared in Example 1 of the present application; FIG. 1B is a SEM image of the carbon-inorganic oxide hybrid micro-nano fiber prepared in Example 1 of the present application;

[0038] FIG. 2A is a photograph of the three-dimensional scaffold prepared in Example 1 of the present application; FIG. 2B is a SEM image of the three-dimensional scaffold prepared in Example 1 of the present application;

[0039] FIG. 3A is an infrared imaging image of the dry state of the three-dimensional scaffold prepared in Example 1 of the present application after laser irradiation; FIG. 3B is an infrared imaging image of the wet state of the three-dimensional scaffold prepared in Example 1 of the present application;

[0040] FIG. 4A is a colony growth image of the three-dimensional scaffold of Example 1 of the present application without irradiation; FIG. 4B is a colony number image of the three-dimensional scaffold of Example 1 of the present application with irradiation, FIG. 4C is a picture under a microscope of FIG. 4A, and FIG. 4D is a picture under a microscope of FIG. 4B;

[0041] FIG. 5A and FIG. 5B show the drug release curve of the three-dimensional scaffold of Example 1 of the present application, wherein FIG. 5A is the drug release curve of the pure carrageenan scaffold, and FIG. 5B is the drug release curve of the hybrid fiber / carrageenan composite three-dimensional scaffold;

[0042] FIG. 6 is a tumor cell activity image of the irradiation group and the non-irradiation group;

[0043] FIG. 7 is a picture of the three-dimensional scaffold of Example 1 of the present application after being soaked in a mineralization solution for one week;

[0044] FIG. 8 is a CT image of the repair of a defect in a rat skull after implanting the three-dimensional scaffold of Example 1 of the present application for 12 weeks;

[0045] FIG. 9 is a flow chart of the preparation method of the three-dimensional scaffold. DETAILED DESCRIPTION

[0046] The following are specific embodiments of the present application, and the technical solutions of the present application are further described in conjunction with the embodiments. However, the present application is not limited to these embodiments.

[0047] The reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field unless otherwise specified.

[0048] Embodiment 1

[0049] The present embodiment provides a three-dimensional scaffold, and the raw materials for preparing the three-dimensional scaffold include carbon-inorganic oxide hybrid micro-nano fibers and carrageenan. The preparation method is shown in FIG. 9, and the specific process is as follows:

[0050] S1, using ethyl silicate as a silicon source, triethyl phosphate as a phosphorus source, ethanol as a solvent, and calcium nitrate tetrahydrate as a calcium source, 11.2 g, 0.72 g, 3.45 g, 20 g, 3 g, and 1 g of the above chemicals were mixed and stirred on a magnetic stirrer for 2 hours to obtain a precursor solution. A template polymer solution was prepared using 10% polyvinyl butyral (PVB). The template polymer solution and the precursor solution were mixed at a ratio of 1:1 and stirred on a magnetic stirrer for 6 hours to ensure uniform mixing, and a spinning solution was obtained.

[0051] S2, the spinning solution in step S1 was placed in a syringe, and electrospinning was performed using an electrospinning device. The obtained precursor micro-nano fibers were received on a substrate. The spinning voltage was 20 kV, the receiving distance was 15 cm, the infusion speed was 1 mL / h, the temperature was 25±5℃, and the relative humidity was 30±5%.

[0052] S3, the obtained precursor micro-nano fibers were placed in a tube furnace for carbonization. The carbonization temperature was 600℃, and the carbonization time was 4 hours. Carbon-inorganic oxide hybrid micro-nano fibers were obtained.

[0053] S4, 0.15 g of carbon-inorganic oxide hybrid nanofibers and 10 mL of 1% carrageenan solution were mixed, and a digital high-speed dispersion homogenizer was used to disperse and uniformly disperse the mixture. The dispersed mixture was divided into 2 mL centrifuge tubes and 1 cm 3 plastic cubic boxes. The centrifuge tubes or cubic boxes were placed in a -80℃ refrigerator for freezing, and the freezing time was 30 minutes. After taking out, it was placed in a freeze dryer, and a vacuum pump was used to extract air. The sample was freeze-dried for 24 hours. The three-dimensional scaffold slices loaded in the 2 mL centrifuge tubes were cut into small round pieces with a height of 2-3 mm to ensure the flatness of the cutting surface, and then the sample was divided into small pieces.

[0054] Figure 1A is a photograph of carbon-inorganic oxide hybrid micro-nanofibers prepared in Example 1, and Figure IB is an SEM image of the carbon-inorganic oxide hybrid micro-nanofibers prepared. Figure 2A is a photograph of a three-dimensional scaffold prepared in Example 1, and Figure 2B is an SEM image of the three-dimensional scaffold prepared in Example 1. The three-dimensional scaffold has a macroporous structure of 30-500 μm, a fiber structure of 200 nm-3,000 nm, and a porosity of 70%-98%.

[0055] Example 2

[0056] This example provides a three-dimensional scaffold, the components and preparation method of which are substantially the same as in Example 1, except that the mass ratio of the template polymer solution and the precursor solution is 1:3.

[0057] The carbon-inorganic oxide hybrid micro-nanofibers prepared in Example 2 were observed using a scanning electron microscope to determine the surface morphology, and were compared with the fibers prepared in Example 1. As the proportion of the template polymer decreased, the carbon content of the hybrid fibers after carbonization decreased, the color of the fiber sample became lighter, but the flexibility increased slightly.

[0058] Example 3

[0059] This example provides a three-dimensional scaffold, the components and preparation method of which are substantially the same as in Example 1, except that the template polymer is polyvinyl alcohol (PVA), and the pre-oxidation temperature is 300°C and the pre-oxidation time is 2 hours.

[0060] Example 4

[0061] This example provides a three-dimensional scaffold, the components and preparation method of which are substantially the same as in Example 1, except that the 10 mL of 1% carrageenan solution was changed to 10 mL of 2% carrageenan solution. As the content of carrageenan increased, the drug release time of the three-dimensional scaffold of Example 4 was longer than that of the three-dimensional scaffold of Example 1.

[0062] Example 5

[0063] This example provides a three-dimensional scaffold, the components and preparation method of which are substantially the same as in Example 1, except that the polymer template is polyvinylpyrrolidone (PVP), the polymer concentration is 8%, and the spinning receiving distance is 20 cm.

[0064] Example 6

[0065] This example provides a three-dimensional scaffold, the components and preparation method of which are substantially the same as in Example 1, except that n-butyl phosphate is used instead of triethyl phosphate.

[0066] Example 7

[0067] The embodiment provides a three-dimensional scaffold, the components and the preparation method of which are basically same with those of the embodiment 1, and the difference is that the electrostatic spinning machine voltage is set to 22KV, the temperature is replaced by 30±5℃, and the humidity is 35±5%.

[0068] Embodiment 8

[0069] The embodiment provides a three-dimensional scaffold, the components and the preparation method of which are basically same with those of the embodiment 1, and the difference is that the equal amount of tetraethyl orthosilicate is used to replace the ethyl silicate.

[0070] Performance test

[0071] The three-dimensional scaffold prepared in the embodiment 1 is subjected to photothermal effect determination, and pure carrageenan scaffolds are used as a control, and the two groups of samples are placed under 1.6W infrared light. FIG. 3A is a dry-state infrared image, and FIG. 3B is a wet-state infrared image, the highest temperature of the three-dimensional scaffold of the embodiment 1 of the application reaches 120℃ in the dry state, the highest temperature of the pure carrageenan control group without fibers is only 39℃ under the infrared light irradiation, the highest temperature of the three-dimensional scaffold in the wet state reaches 73℃, and the pure carrageenan is 33℃.

[0072] Further, the three-dimensional scaffold of the embodiment 1 is subjected to antibacterial performance test, 8 scaffold samples (4 groups of irradiation and non-irradiation) are prepared and placed in 300 microliters of staphylococcus aureus liquid. The samples in the irradiation group are subjected to irradiation treatment before being placed, to ensure that the samples are irradiated by the infrared light. The samples in the non-irradiation group are directly placed in the staphylococcus aureus liquid without light treatment. Then the liquid is diluted by 10 times, mixed with the culture medium and placed in a 37℃ constant temperature incubator. The colony generation in the culture dish is observed. FIG. 4A is a colony growth diagram without irradiation, FIG. 4B is a colony growth diagram after irradiation, FIG. 4C is a picture under a microscope of FIG. 4A, and FIG. 4D is a picture under a microscope of FIG. 4B. It can be seen from the pictures under the microscope that the number of colonies in the culture dish under the infrared light irradiation is obviously reduced.

[0073] Further, the three-dimensional scaffold of the embodiment 1 is subjected to drug absorption performance determination, 5mL of methotrexate solution with a concentration of 0.05mg / mL is taken. 0.1g of hybrid nanofiber is mixed with the methotrexate solution, and is fully stirred to be uniform, so that the drug and the nanofiber are subjected to adsorption reaction. At different time points in the adsorption process, a fixed volume of clear solution (without fiber) is taken, and the absorption light value is measured by using an ultraviolet spectrophotometer. The absorption light value at each time point is recorded. According to the absorption light value obtained in the last step, the drug loading of methotrexate is calculated by using a regression equation. The final adsorption amount of methotrexate is 2.052mg / g, and the adsorption rate is 80.2%.

[0074] Further, the three-dimensional scaffold of Example 1 was subjected to drug release performance determination, and four composite drug-loaded scaffolds with a diameter of 6 mm and a height of 2 mm and pure carrageenan scaffolds were prepared. The two groups of scaffolds were placed in PBS solution, and the water bath was set to 37℃. The sample solution was collected at regular intervals within 12 hours, and the ultraviolet absorbance value at each time point was measured. The drug release curve was plotted, as shown in FIGS. 5A and 5B. The three-dimensional scaffold of Example 1 reached drug release equilibrium in 7 hours, while the drug release of pure carrageenan reached release equilibrium in 1.5 hours.

[0075] Further, human osteosarcoma cells (U2OS) were planted in the three-dimensional scaffold of Example 1, and after a period of culture, the scaffold was irradiated with infrared laser to kill the tumor cells. As shown in FIG. 6, the cell activity of the scaffold after laser irradiation was significantly less than that of the non-irradiation group, indicating that the scaffold has good anti-tumor effect.

[0076] Further, a three-dimensional scaffold of Example 1 was taken, and a 4 cm 2 The sample was placed in a mineralization solution for immersion for one week, as shown in FIG. 7. After immersion, the fibers showed obvious mineral crystallization, indicating that the three-dimensional scaffold has good bioactivity.

[0077] Further, the scaffold after freeze-drying was made into a composite drug-loaded scaffold with a diameter of 6 mm and a height of 2 mm for treatment of mouse skull defects. As shown in FIG. 8, after twelve weeks, CT observation of the mouse skull found that the mouse head had obvious bone growth, proving that the carbon-inorganic nanofiber has good osteogenic repair ability and better osteogenic activity, and can be used as a bone tissue regeneration scaffold for application in bone tissue repair.

[0078] The above is described in detail in combination with the embodiments of the present application, but the present application is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the purpose of the present application.

Claims

1. A three-dimensional scaffold, characterized in that, The preparation raw material of the three-dimensional scaffold includes carbon-inorganic oxide hybrid micro-nano fibers and carrageenan.

2. The three-dimensional scaffold of claim 1, wherein, The three-dimensional scaffold has a macroporous structure of 30-500 μm, a fiber structure of 200 nm-3000 nm, and a porosity of 70%-98%.

3. The three-dimensional scaffold of claim 1, wherein, The carbon-inorganic oxide hybrid micro-nano fibers are prepared by the following method: S1, mixing and stirring a silicon source, a phosphorus source, ethanol, a calcium source, an acid catalyst, and water to obtain a precursor solution, mixing a template polymer solution and the precursor solution to obtain a spinning solution; S2, electrospinning the spinning solution, and then carbonizing to obtain the carbon-inorganic oxide hybrid micro-nano fibers.

4. The three-dimensional scaffold of claim 3, wherein, The mass ratio of the template polymer solution and the precursor solution is 1:10-1:0.

1.

5. The three-dimensional scaffold of claim 3, wherein, In step S2, the parameters of the electrospinning are as follows: The spinning voltage is 10-30 kV, the receiving distance is 5-30 cm, the perfusion speed is 0.5-5 mL / h, the temperature is 15-35 °C, and the relative humidity is 25%-60%.

6. The three-dimensional scaffold of claim 3, wherein, In step S2, before the carbonization, a pre-oxidation step is further included.

7. The three-dimensional scaffold of claim 3, wherein, In step S2, the carbonization temperature is 200-800 °C.

8. The method of producing a three-dimensional scaffold according to any one of claims 1 to 7, wherein The method includes the following steps: (1) mixing the carbon-inorganic oxide hybrid micro-nano fibers, the carrageenan, and water, and uniformly dispersing to obtain a mixture; (2) freezing the mixture, and then freeze-drying to obtain the three-dimensional scaffold.

9. The method of claim 8, wherein the three-dimensional scaffold is prepared by, The mass ratio of the carbon-inorganic oxide hybrid micro-nano fibers and the carrageenan is 1:(0.1-10).

10. Use of the three-dimensional scaffold according to any one of claims 1-7 in the preparation of a medical material for treating osteosarcoma.

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