A bionic three-dimensional nanoscaffold for promoting the repair of aging bone defects and its preparation method

Abstract

The present invention provides a bionic three-dimensional scaffold for promoting the repair of aged bone defects and targeting the aging pathway, and a preparation method thereof, the method comprising the following steps: 1) preparing mesoporous bioactive glass nanospheres; 2) loading anti-aging drugs or inhibitors targeting the aging pathway into the mesoporous bioactive glass nanospheres; 3) blending the drug-loaded nanospheres with polycaprolactone to obtain a nanoscale fiber membrane; 4) cutting the fiber membrane into pieces, freeze-drying to obtain a drug-loaded three-dimensional electrospinning nanoscaffold; 5) placing in a polydopamine solution for surface modification. A bionic three-dimensional scaffold for promoting the repair of aged bone defects and targeting the aging pathway provided by the present invention has an extracellular matrix structure similar to that of natural bone, and the loaded drugs can target the aging mechanism, reduce the burden of aging cells, and increase cell activity, and can be used for the repair of aged bone defects. It can also prevent multiple aging complications at the same time by targeting the basic aging mechanism, providing a direction for future clinical trials.

Description

Technical Field

[0001] The present invention relates to the fields of nanomaterials and biological tissue engineering, and more specifically to a bionic three-dimensional scaffold for promoting the repair of aged bone defects by targeting an aging pathway and a preparation method thereof. Background Art

[0002] As we age, bone strength decreases, and bone diseases such as fractures and osteoporosis become increasingly serious health problems. Given that age-related bone diseases often coexist with a variety of other chronic diseases such as atherosclerosis, diabetes, and frailty, there is growing interest in the "gerontology hypothesis," which posits that manipulation of basic aging mechanisms will delay the onset or severity of a variety of chronic diseases that often have aging as an underlying risk factor. Therefore, targeting basic aging mechanisms could help treat a variety of age-related diseases.

[0003] The aging of the body is associated with the increase of senescent cells. Senescent cells gradually accumulate in bone tissue with age, leading to the loss of self-repair ability of bone tissue and the occurrence of dysfunction. Senescent cells not only highly express biomarkers p16, p21, and p53, leading to increased expression of cyclin kinase inhibitors, but also resist apoptosis. In addition, the senescence-associated exocrine phenotype (SASP) IL-6, IL-8, MMP13, PAI-1, etc. of senescent cells will affect the body's function. With the increase of age, various cells in the aged bone microenvironment will age, the ability of bone marrow stem cells to form osteoblasts will decrease, and adipocytes will be formed preferentially, leading to reduced bone formation and mineralization. Some drug molecules can inhibit aging-related phenotypes by specifically inhibiting SASP or proinflammatory factors. For example, anti-aging drugs: anti-aging drugs include dasatinib + quercetin or kinase inhibitors that inhibit the production of aging-related exocrine proinflammatory factors.

[0004] Designing bone-mimetic scaffolds involves constructing appropriate bone representations, which requires an understanding of bone biology and physiology. Bone can be viewed as a composite material consisting of osteoblast extracellular matrix (ECM) proteins, growth factors, mineral calcium in the form of calcium hydroxyapatite, and a complex vascular system. Traditionally, scaffolds have been designed to resemble the macroscopic structure of natural tissues and organs, however, little attention has been paid to the complexity and nanoscale details of the microscopic structure of tissues and organs. Summary of the invention

[0005] The purpose of the present invention is to provide a bionic three-dimensional nano-scaffold that promotes the repair of aged bone defects and targets the aging pathway, and a preparation method thereof, thereby solving the problem in the prior art of lacking a bone bionic scaffold that effectively improves the quality and strength of aged bones.

[0006] To solve the above problems, the present invention adopts the following technical solutions:

[0007] Provided is a method for preparing a biomimetic three-dimensional scaffold that targets an aging pathway and promotes the repair of aged bone defects, comprising the following steps: 1) using a small molecule cationic surfactant CTAB and polystyrene-b-polyacrylic acid micelles as structure directing agents, adding tetraethyl orthosilicate and calcium nitrate as silicon sources and calcium sources, and preparing mesoporous bioactive glass nanospheres with surface-exposed macropores and high specific surface area; 2) dispersing the mesoporous bioactive glass nanospheres obtained in step 1) in an anti-aging drug solution, stirring at room temperature in the dark, centrifuging, washing, and drying to obtain drug-loaded mesoporous bioactive glass nanospheres; 3) dissolving polycaprolactone particles in a hexafluoroisopropanol solvent and stirring evenly to obtain a polycaprolactone solution with a mass fraction of 7%-15%, and dispersing the drug-loaded mesoporous bioactive glass nanospheres obtained in step 2) in a solution of an anti-aging drug. The rice balls are dispersed in a polycaprolactone solution and transferred to a syringe, and an electrospinning technology is used to prepare a fiber membrane containing bioactive nanospheres. The process conditions of electrospinning are: a flow rate of 0.1-0.3 mL / h, a voltage of 15-18 KV, and a receiving distance of 10-15 cm; 4) the fiber membrane obtained in step 3) is placed in a glutaraldehyde steam box for cross-linking, cut into pieces and dispersed in deionized water, and placed in a homogenizer to break into a uniform short nanofiber suspension, and then the short nanofiber suspension is placed in a mold and freeze-dried to form a nanoscale three-dimensional electrospinning scaffold; 5) the nanoscale three-dimensional electrospinning scaffold obtained in step 4) is surface modified using polydopamine to obtain a bionic three-dimensional scaffold that targets the aging pathway and promotes the repair of aged bone defects.

[0008] According to the present invention, a method for preparing a bionic three-dimensional nanoscaffold that targets the aging pathway and promotes the repair of aged bone defects is provided. The main structure of the scaffold is made into a nanoscale scaffold by electrospinning a blend of polycaprolactone biomaterial and mesoporous bioactive glass nanospheres, wherein the mesoporous bioactive glass nanospheres can be loaded with anti-aging drugs or inhibitors that inhibit the production of SASP, such as JAKi. On the one hand, they simulate the organic and inorganic structure of natural bones, and on the other hand, they target senescent cells, thereby reducing the burden of senescent cells, promoting aged bone repair, and achieving improvement in the quality and strength of aged bones.

[0009] Preferably, in step (1), polystyrene-b-polyacrylic acid is dissolved in tetrahydrofuran, and the mixture is quickly poured into an alkaline solution containing CTAB to obtain dual-template composite micelles as a structure directing agent for mesoporous bioactive glass nanospheres. Then, tetraethyl orthosilicate and calcium nitrate tetrahydrate are added and stirred, centrifuged and collected, vacuum dried, and then calcined in a muffle furnace to prepare mesoporous bioactive glass nanospheres with surface-exposed macropores and high specific surface area. The preparation method can regulate the material morphology and pore size. The mesoporous bioactive glass nanospheres prepared by the present invention have excellent biological activity on the one hand, and can be evenly distributed on polycaprolactone fibers to simulate the combination of collagen fibers and hydroxyapatite in natural bones. On the other hand, they can be used as carriers to load and deliver anti-aging drugs.

[0010] Preferably, in step 1), the molar ratio of tetraethyl orthosilicate to calcium nitrate is (75-85): (25-15), which can obtain a good spherical morphology and pore size, preferably with a pore size range of 10nm-20nm, a calcination temperature of 500-600°C, and a heating rate of 1-1.5°C / min.

[0011] The anti-aging drug in step 2) can be an anti-aging drug molecule: including dasatinib + quercetin, navitoc, piperamide, etc., or a kinase inhibitor that inhibits the production of aging-related exocrine pro-inflammatory factors: ruxolitinib, tofacitinib, etc.

[0012] The amount of drug-loaded nanospheres added in step 3) is 1-2% (g / 100 mL). After stirring evenly, the mixture is allowed to stand until the bubbles in the solution disappear completely.

[0013] In step 4), the fiber membrane is broken at a speed of 8000-12000 rpm. After multiple observations, the fiber membrane is completely broken into short fibers.

[0014] In step 4), the electrospinning technology and the freeze-drying technology are combined to freeze-dry the electrospun nanocomposite short fibers to obtain a three-dimensional porous scaffold with a pore size of 10-300 μm and a porosity of >90%.

[0015] In order to improve the hydrophilicity of the scaffold surface and thus improve cell adhesion, in step 5), the freeze-dried three-dimensional scaffold is placed in a dopamine hydrochloric acid solution for 8-12 hours and then rinsed with deionized water several times to remove unreacted dopamine.

[0016] It is well known that for elderly bone defects, intravenous injection or oral medication is usually difficult to reach the local defect site. For the repair of elderly bones, the requirements for three-dimensional scaffolds are higher, not only to simulate the macro structure, but also to simulate the micro-nano structure of natural bones. For the repair of elderly bones, since its complex microenvironment is not conducive to cell proliferation, combining nanostructures and biological growth factors or drugs into scaffolds is the most valuable and challenging treatment method.

[0017] The main inventive point of the present invention is that it combines the targeted aging pathway mechanism with a three-dimensional scaffold for the first time. By combining nano-scale polycaprolactone fibers with nano-bioactive glass, the contact sites of cells in the aged bone microenvironment are effectively increased, providing cells with a suitable adhesion and proliferation space. Furthermore, by in situ delivery of anti-aging drugs, the aged bone microenvironment is improved, the burden of aging cells is reduced, thereby achieving the effect of promoting aged bone repair.

[0018] It should be understood that the present invention uses polycaprolactone to construct a scaffold, and the nanoscale scaffold that simulates the natural bone cell matrix structure provides more binding sites for cells, targets the binding of aging drugs or inhibitors to inhibit the production of SASP, and reduces the burden on mesenchymal bone marrow stem cells in the aging bone microenvironment, thereby increasing their activity and promoting the repair of aged bones. This strategy is fundamentally different from all current drugs for treating aging bone diseases.

[0019] Compared with the prior art, the bionic three-dimensional nanoscaffold provided by the present invention for promoting the repair of aged bone defects and targeting the aging pathway has the following beneficial effects:

[0020] 1) The bionic three-dimensional scaffold for promoting the repair of aged bone defects and targeting the aging pathway provided by the present invention has a nanoscale structure, and the drug-loaded mesoporous bioactive glass is uniformly dispersed in a single nanoscale fiber, simulating the organic and inorganic components in natural bone, and has an extracellular matrix structure similar to that of natural bone. The loaded drugs can target the aging mechanism, reduce the burden of senescent cells, and thus improve cell activity for the repair of aged bone defects.

[0021] 2) The present invention adopts a treatment method that combines targeted aging pathway mechanisms with bone repair scaffolds, not only targeting bone-specific pathways, but also targeting basic aging mechanisms present in all tissues. The bionic three-dimensional nanoscaffold can release anti-aging drugs in situ to target senescent cells, inhibit the production of toxic substances SASP, reduce the burden of senescent cells, and thus promote bone tissue repair; at the same time, inhibiting the production of SASP in senescent cells has been shown to improve cardiovascular function, enhance insulin sensitivity, and alleviate weakness, making it fundamentally different from all current treatments for aging bone diseases (only beneficial to bones). Therefore, such a bone repair scaffold for senescent cell treatment provided by the present invention is not only a new strategy for treating aging bone diseases, but also can simultaneously prevent multiple aging complications by targeting basic aging mechanisms, providing a direction for future clinical trials. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 A design flow chart of a bionic three-dimensional scaffold for promoting the repair of aged bone defects that targets the aging pathway provided by the present invention;

[0023] Figure 2 This is a scanning electron microscope image of mesoporous bioactive glass nanospheres;

[0024] Figure 3 This is the surface scanning electron microscope image of the nanofiber membrane;

[0025] Figure 4 The diameter distribution diagram of the fiber membrane containing bioactive nanospheres prepared by electrospinning technology;

[0026] Figure 5 This is a scanning electron microscopy image of the nanoscale three-dimensional electrospun scaffold obtained after freeze drying;

[0027] Figure 6 This is a digital photo of a nanoscale three-dimensional electrospun scaffold;

[0028] Figure 7 This is a digital photo of a biomimetic three-dimensional scaffold modified with polydopamine. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0030] According to the present invention, a method for preparing a bionic three-dimensional scaffold for promoting the repair of aged bone defects by targeting the aging pathway is provided, and the method comprises the following steps: 1) preparing mesoporous bioactive glass nanospheres; 2) loading anti-aging drugs or inhibitors targeting the aging pathway into the mesoporous bioactive glass nanospheres; 3) blending the drug-loaded nanospheres with polycaprolactone to obtain a nanoscale fiber membrane; 4) cutting the fiber membrane into pieces, freeze-drying it to obtain a drug-loaded three-dimensional electrospun nanoscaffold; 5) placing it in a dopamine solution for surface modification. The overall design process is as follows: Figure 1 shown.

[0031] Example 1

[0032] This embodiment provides a method for preparing a bionic three-dimensional scaffold that targets an aging pathway and promotes the repair of aged bone defects, comprising the following steps:

[0033] (1) Add 50 mg of PS-b-PAA powder to 10 mL of tetrahydrofuran solution, stir and dissolve at room temperature, then quickly pour it into an alkaline solution containing CTAB (add 50 mg of CTAB and 0.5 mL of ammonia water to 20 mL of ultrapure water), then stir the mixed solution in a 35°C water bath for 30 min, add tetraethyl orthosilicate and calcium nitrate tetrahydrate (Si:Ca 75:25) in turn and stir overnight, collect the obtained sample by centrifugation (10000 rpm, 10 min), wash three times with ethanol and ultrapure water respectively. After vacuum drying, place it in a muffle furnace and calcine it at 550°C for 6 h (heating rate of 1°C / min) to obtain mesoporous bioactive glass nanospheres. Figure 2 As shown, the mesoporous bioactive glass nanospheres have a regular spherical morphology and exposed macroporous structure, and the pore size is about 150 nm.

[0034] (2) A 0.1 mg / mL drug solution is prepared with PBS buffer (pH=7.4), 10 mg of the mesoporous bioactive glass nanospheres in step (1) are weighed, stirred at room temperature in the dark for 24 h, and then the sample is collected by centrifugation (10000 rpm, 10 min), washed with PBS several times, and then dried in a vacuum drying oven to obtain drug-loaded mesoporous bioactive glass nanospheres. The drug solution can be an anti-aging drug or a kinase inhibitor that inhibits the production of aging-related exocrine pro-inflammatory factors.

[0035] (3) Place the polycaprolactone particles in 10 mL of hexafluoroisopropanol and stir at room temperature to form a clear solution to prepare a 10% mass fraction polycaprolactone solution. Disperse the drug-loaded mesoporous bioactive glass nanospheres prepared in step (2) in the above-mentioned clear solution and stir for 2 hours to make them evenly dispersed. Then place the mixed solution at room temperature to remove the bubbles therein, transfer it into a syringe, and fix a 23G syringe needle with a flattened end at the syringe port as the spinneret for electrospinning. The process conditions of the electrospinning are: flow rate of 0.3 mL / h, voltage of 15 KV, and receiving distance of 15 cm. Thereby, a nanofiber membrane with evenly dispersed mesoporous bioactive glass nanospheres is obtained. Figure 3 As shown in Figure 2, the mesoporous bioactive glass nanospheres are embedded and filled in the nanofiber matrix along the axial direction of the fiber. The individual fibers are randomly dispersed, loosely overlapped and interwoven, and the fiber diameters are evenly distributed, about 130 nm (e.g. Figure 4 shown).

[0036] (4) The nanofiber membrane obtained in step (3) was peeled off from the collecting table and placed in a glutaraldehyde steam box for crosslinking for 2 hours, then cut into 1 cm × 1 cm fiber pieces and dispersed in deionized water, and then placed in a homogenizer to break into a uniform short nanofiber suspension. The suspension was then placed in a cylindrical mold and freeze-dried for 24 hours to form a nanoscale three-dimensional electrospun scaffold, the scanning electron microscope image of which is shown in FIG. Figure 5 As shown in the figure, the three-dimensional nanofiber morphology can be seen, which has interconnected pores of different sizes, ranging from tens of microns to hundreds of microns. This result shows that the fiber morphology has not changed after cross-linking and freeze-drying. The digital photo of the nanoscale three-dimensional electrospun scaffold is shown in Figure 6 shown.

[0037] (5) The scaffold obtained in step (4) is surface-modified with polydopamine to improve the hydrophilicity of the scaffold surface. The nanofiber scaffold is immersed in a dopamine hydrochloric acid solution (2 mg / mL, 10 mM Tris-HCl buffer, pH = 8.5) for 12 h, and then thoroughly washed with deionized water several times to remove excess unreacted dopamine molecules. After vacuum drying, a polydopamine-modified biomimetic three-dimensional scaffold is finally obtained, such as Figure 7 shown.

[0038] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiments of the present invention can also be modified in various ways. All simple, equivalent changes and modifications made according to the claims and the description of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A method for preparing a bionic three-dimensional scaffold for promoting the repair of aged bone defects by targeting the aging pathway, characterized in that: The following steps are involved: 1) The small molecule cationic surfactant CTAB and polystyrene-b-polyacrylic acid micelles were used as structure directing agents, and tetraethyl orthosilicate and calcium nitrate were added as silicon source and calcium source to prepare mesoporous bioactive glass nanospheres with surface exposed macropores and high specific surface area; 2) dispersing the mesoporous bioactive glass nanospheres obtained in step 1) in an anti-aging drug solution, stirring at room temperature in the dark, centrifuging, washing, and drying to obtain drug-loaded mesoporous bioactive glass nanospheres; 3) dissolving polycaprolactone particles in hexafluoroisopropanol solvent and stirring evenly to obtain a polycaprolactone solution with a mass fraction of 7%-15%, dispersing the drug-loaded mesoporous bioactive glass nanospheres obtained in step 2) in the polycaprolactone solution, and transferring them to a syringe, and preparing a fiber membrane containing bioactive nanospheres by electrospinning technology, wherein the process conditions of electrospinning are: flow rate of 0.1-0.3mL / h, voltage of 15-18KV, and receiving distance of 10-15cm; 4) placing the fiber membrane obtained in step 3) in a glutaraldehyde steam box for cross-linking, cutting it into pieces and dispersing it in deionized water, placing it in a homogenizer to break it into a uniform short nanofiber suspension, and then placing the short nanofiber suspension into a mold, freeze-drying it to form a nanoscale three-dimensional electrospinning scaffold; 5) Using polydopamine to modify the surface of the nanoscale three-dimensional electrospinning scaffold obtained in step 4) to obtain a bionic three-dimensional scaffold that targets the aging pathway and promotes the repair of aged bone defects.

2. The preparation method according to claim 1, characterized in that: In the step 1), polystyrene-b-polyacrylic acid is first dissolved in tetrahydrofuran, and then quickly poured into an alkaline solution containing CTAB to obtain dual-template composite micelles, and then added with tetraethyl orthosilicate and calcium nitrate tetrahydrate, stirred, centrifuged, collected, vacuum dried, and then calcined in a muffle furnace.

3. The preparation method according to claim 2, characterized in that: In the step 1), the molar ratio of tetraethyl orthosilicate to calcium nitrate is (75-85): (25-15), the calcination temperature in the muffle furnace is 500-600° C., and the heating rate is 1-1.5° C. / min.

4. The preparation method according to claim 1, characterized in that: In the step 2), the anti-aging drug is an anti-aging drug molecule or a kinase inhibitor that inhibits the production of aging-related exocrine pro-inflammatory factors.

5. The preparation method according to claim 4, characterized in that: In the step 2), the anti-aging drug molecules include: dasatinib + quercetin, navitoc, piperamide, and the kinase inhibitors include: ruxolitinib, tofacitinib.

6. The preparation method according to claim 1, characterized in that: In the step 3), the amount of the drug-loaded mesoporous bioactive glass nanospheres added is 1%-2%, and the unit of the amount added is g / 100mL. After stirring evenly, the solution is allowed to stand until the bubbles in the solution completely disappear.

7. The preparation method according to claim 1, characterized in that: In the step 4), the fiber membrane is broken at a rotation speed of 8000-12000 rpm, so that the fiber membrane is completely broken into short fibers.

8. The preparation method according to claim 1, characterized in that: In the step 4), a nanoscale three-dimensional electrospinning scaffold with a pore size of 10-300 μm and a porosity of >90% is obtained by freeze drying.

9. The preparation method according to claim 1, characterized in that: In step 5), the freeze-dried nanoscale three-dimensional electrospun scaffold is placed in a dopamine hydrochloric acid solution for 8-12 hours, and then rinsed with deionized water several times to remove unreacted dopamine.

10. A bionic three-dimensional scaffold targeting the aging pathway and promoting the repair of aged bone defects, prepared according to the preparation method according to any one of claims 1 to 9.

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