A pterygoid sinus support and a preparation method and application thereof

Abstract

The application discloses a mastoid air cell stent and a preparation method and application thereof, and belongs to the technical field of biomedical materials. The mastoid air cell stent comprises a biodegradable solid stent, wherein the solid stent has air cell pores; further comprises biodegradable hydrogel, wherein the hydrogel comprises liposomes, the liposomes load drugs, the drugs comprise one or a combination of pro-angiogenic drugs, pro-osteogenic drugs and anti-inflammatory drugs; the hydrogel is at least partially filled in the air cell pores; and / or, at least partially coated on the surface of the solid stent. The mastoid air cell stent has a "soft package hard" structure that the hydrogel coats the solid stent with a bionic air cell pore structure, the drug-loaded hydrogel promotes angiogenesis and bone repair, the solid stent promotes new bone formation air cell, and thus the mastoid air cell reconstruction is realized and the air pressure regulation function is recovered.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a mastoid air cell stent, its preparation method, and its application. Background Technology

[0002] Chronic suppurative otitis media (CSOM) and middle ear cholesteatoma (MEC) are common ear diseases in China. When the disease progresses to the middle or late stages, surgery becomes the primary treatment, but a certain recurrence rate exists post-surgery, which is closely related to the imbalance of air pressure within the middle ear. Normally, the air pressure in the middle ear is balanced by the connection between the mastoid air cells, the tympanic cavity, and the Eustachian tubes. Once this balance is disrupted, negative pressure in the ear can easily occur, leading to a series of ear diseases. Most mastoid cells have a honeycomb-like structure, interconnected with each other, increasing the air-containing space in the middle ear and thus acting as a pressure buffer. However, during CSOM and MEC surgery, it is often necessary to remove the damaged mastoid tissue. The commonly used simple packing method cannot restore the original structure of the mastoid air cells, causing them to lose their pressure regulation function, potentially leading to middle ear pressure imbalance and disease recurrence. Therefore, how to reconstruct the mastoid air cells, restore their pressure regulation function, eliminate the potential conditions for disease recurrence, and thus overcome the treatment bottleneck has become a crucial problem that urgently needs to be solved in otology. Summary of the Invention

[0003] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, one objective of this invention is to provide a mastoid air cell stent, which has a "soft-wrapped-hard" structure of a solid scaffold with a biomimetic air cell pore structure encapsulated by hydrogel, simultaneously achieving structural support and functional repair. The drug-loaded hydrogel promotes angiogenesis and bone repair, while the solid scaffold promotes new bone formation of air cells, thereby achieving mastoid air cell reconstruction and restoring its baroregulation function. A second objective of this invention is to provide a method for preparing the mastoid air cell stent described in the first aspect. A third objective of this invention is to provide an application of the mastoid air cell stent described in the first aspect.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention is to provide a mastoid air cell stent, comprising a biodegradable solid stent having air cell pores; It also includes biodegradable hydrogels containing liposomes loaded with drugs, the drugs including one or a combination of angiogenic drugs, osteoproliferative drugs, and anti-inflammatory drugs. The hydrogel at least partially fills the pores of the air chamber; and / or at least partially covers the surface of the solid scaffold.

[0005] The mastoid air cell scaffold of this invention has a "soft-wrapped-hard" structure, in which a solid scaffold with a biomimetic air cell pore structure is covered by hydrogel. The liposome-loaded drug-eluting hydrogel releases drugs through degradation to promote angiogenesis and osteoogenesis and inhibit local inflammation. Although the hydrogel soft material degrades relatively quickly, it can be replenished by injection. The degraded area can gradually form the mastoid air cell bone wall. The solid scaffold provides mechanical support by constructing a biomimetic mastoid air cell structure. Its degradation rate is slower than that of the hydrogel soft material. It gradually degrades or physically dissolves to form air cells in the later stage of new bone formation, while releasing drugs to inhibit the proliferation of granulation tissue into the air cells.

[0006] Encapsulating drugs in liposomes can significantly improve their sustained-release properties and bioavailability.

[0007] In some preferred embodiments, the mass ratio of the solid scaffold to the hydrogel is 1:(1-10), more preferably 1:(3-8), and even more preferably 1:(5-6).

[0008] In some preferred embodiments, the concentration of the liposomes in the hydrogel is 0.1-1.0 mg / mL, more preferably 0.3-0.7 mg / mL, and even more preferably 0.4-0.6 mg / mL.

[0009] In some preferred embodiments, the concentration of the liposomes in the solid scaffold is 0.2-10 μg / mg, more preferably 0.5-8 μg / mg, and even more preferably 1-5 μg / mg.

[0010] The concentration of liposomes in the solid scaffold refers to the average concentration of liposomes loaded in the hydrogel filling the pores of the air cells relative to the overall concentration of the solid scaffold.

[0011] In some preferred embodiments, the solid scaffold material comprises a polymer of polycaprolactone and alkenyl poloxamer.

[0012] In some preferred embodiments, the mass ratio of the polymerized alkenyl poloxamer to the polycaprolactone is 1:(0.1-5), more preferably 1:(0.5-3).

[0013] The scaffold material composed of the polymer products of polycaprolactone and alkenyl poloxamer combines the characteristics of the polymer products of polycaprolactone and alkenyl poloxamer, with a moderate degradation rate that is compatible with the rate of new bone formation.

[0014] In some preferred embodiments, the degradation rate can also be controlled by adjusting the composition ratio of the solid scaffold material or its ratio with the hydrogel material.

[0015] The degradation rate is specifically controlled by adjusting the ratio of polycaprolactone and alkenyl poloxamer polymers in the solid scaffold. By adjusting the ratio of alkenyl poloxamer polymers to polycaprolactone within a mass ratio range of 1:(0.5-3), the degradation time of the solid scaffold can be controlled from 80 to 200 days.

[0016] In some preferred embodiments, the hydrogel material comprises a polymer of at least one of alkenyl-modified gelatin, alkenyl-modified hyaluronic acid, and alkenyl-modified sodium alginate.

[0017] In some preferred embodiments, the hydrogel material comprises a polymer of alkenyl-modified gelatin.

[0018] Gelatin possesses excellent biocompatibility, a microporous structure, and a controllable degradation rate. After alkenyl modification, the natural arginine-glycine-aspartic acid (RGD) sequence of gelatin is retained, forming an injectable, photocrosslinkable, and curable hydrogel precursor that exhibits good compatibility with solid scaffolds and cells. The degradation time of the polymerized product of alkenyl modified gelatin is relatively short, approximately 28 days. After degradation, the hydrogel material forms a bone wall, while the solid scaffold degrades to form air cells.

[0019] In some preferred embodiments, the drug comprises deferoxamine mesylate and dexamethasone.

[0020] Deferroamine mesylate (DFO) can promote vascularized bone repair by inhibiting the degradation of Hif-1α; dexamethasone (DEX) can inhibit the inflammatory response and synergistically promote normal angiogenesis and bone formation with DFO.

[0021] In some preferred embodiments, the concentration of deferoxamine mesylate in the liposomes is 5-20 μg / mg, more preferably 8-15 μg / mg.

[0022] In some preferred embodiments, the concentration of dexamethasone in the liposomes is 0.5-20 μg / mg, more preferably 1-15 μg / mg.

[0023] A second aspect of the present invention is to provide a method for preparing the mastoid air cell stent described in the first aspect, comprising the following steps: (1) Prepare a solid scaffold with air cell pores according to the pore structure of the mastoid air cells; (2) Preparation of drug-loaded liposomes; (3) The solid scaffold is placed in a solution containing the liposomes for swelling equilibrium; (4) The hydrogel precursor solution containing liposomes is placed in a mold containing a solid scaffold after swelling equilibrium and solidified to obtain the papillary air cell scaffold.

[0024] In some preferred embodiments, the solid scaffold is fabricated by 3D printing.

[0025] In some preferred embodiments, the 3D printing method includes the following steps: First, the target air cell structure is obtained, which can be obtained through CT scans or other medical imaging methods. The air cell pores of the solid scaffold are designed according to the obtained target air cell structure. The design structure is encoded into a language that the 3D printer can recognize, and the solid scaffold is printed according to the design structure.

[0026] In some preferred embodiments, the method for preparing the ink for 3D printing includes the following steps: After dissolving the photoinitiator, alkenyl poloxamer and polycaprolactone are added, and the mixture is stirred under light-protected conditions until completely dissolved to obtain ink for 3D printing.

[0027] In some preferred embodiments, the hydrogel precursor solution includes a photoinitiator, a crosslinking monomer, a drug-loaded liposome, and a solvent.

[0028] In some preferred embodiments, the crosslinking monomer includes alkenyl-modified gelatin.

[0029] In some preferred embodiments, the concentration of crosslinking monomers in the hydrogel precursor solution is 10-30 g / 100 mL.

[0030] In some preferred embodiments, the concentration of the liposomes in the hydrogel precursor solution is 0.1-1 mg / mL.

[0031] In some preferred embodiments, the method for preparing the hydrogel precursor solution includes the following steps: After dissolving the photoinitiator, add alkenyl-modified gelatin and stir until completely dissolved under light-protected conditions. Then add the liposome dispersion and mix thoroughly to obtain the hydrogel precursor solution.

[0032] In some preferred embodiments, the liposomes are prepared using a thin-film rotary evaporation-ultrasound method.

[0033] In some preferred embodiments, the thin-film rotary evaporation-ultrasound method includes the following steps: The membrane material constituting the liposomes and the lipid-soluble drug are dissolved in an organic solvent to form a homogeneous solution. The organic solvent is removed by rotary evaporation under reduced pressure to form a liposome membrane. A hydration medium containing a water-soluble drug and small glass beads are added for hydration and washing of the membrane. After ultrasonic treatment, the membrane is dialyzed to obtain the liposomes.

[0034] In some preferred embodiments, the membrane material constituting the liposomes includes egg yolk lecithin and cholesterol.

[0035] A third aspect of the present invention is to provide an application of the mastoid air cell stent described in the first aspect, specifically in promoting angiogenesis or bone repair.

[0036] The beneficial effects of this invention are: (1) The present invention provides a mastoid air cell stent, which has a "soft-covered hard" structure of a solid stent with a biomimetic air cell pore structure covered by hydrogel. It can be used for the reconstruction of mastoid defect air cells. The liposome drug-loaded hydrogel promotes angiogenesis and bone repair, and the solid stent promotes the formation of new bone air cells, thereby realizing the reconstruction of mastoid air cells and restoring their pressure regulation function.

[0037] (2) The present invention also provides a mastoid air cell scaffold with deferroamine mesylate and dexamethasone as drug loading, which is dispersed in hydrogel by liposome encapsulation, significantly improving the sustained release performance and bioavailability of deferroamine mesylate and dexamethasone. At the same time, dexamethasone can also synergistically promote normal angiogenesis and bone formation with deferroamine mesylate.

[0038] (3) The present invention also provides a method for preparing the mastoid air cell stent, wherein the preparation method adopts mold forming, which can simply and efficiently prepare the mastoid air cell stent of the present invention. Attached Figure Description

[0039] Figure 1 This is a photograph of the solid scaffold obtained in Embodiment 1 of the present invention; Figure 2 This is a photograph of the mastoid air cell stent prepared according to Embodiment 1 of the present invention. Figure 3 The in vitro release curves of DFO and DEX under free and liposome loading conditions are shown. Detailed Implementation

[0040] The present invention will be further described in detail below through specific embodiments.

[0041] Unless otherwise specified, the raw materials, reagents, or apparatus used in the following examples and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0042] Embodiments of the present invention relate to a mastoid air cell stent, the preparation method of which includes the following steps: (1) 3D modeling.

[0043] (2) Solid scaffold printing: Dilute 0.05-0.2 g / mL of 2,2-dimethoxy-2-phenylacetophenone (DMPA) solution with acetone to 2-8 mg / mL, add F127DA (poloxam bisacrylamide) and PCL (polycaprolactone) in a mass ratio of 1:(0.5-3) at a material-to-liquid ratio of 1 g / mL, stir for 12-24 h under light-protected conditions until completely dissolved to obtain 3D printing ink, and 3D print according to the structural model in step (1) to obtain a solid scaffold.

[0044] (3) Liposome preparation: 80 mg egg yolk lecithin (EL), 20 mg cholesterol and 0.1-0.3 mg dexamethasone were mixed and dissolved in 4-10 mL of anhydrous ethanol and stirred for 1 h. Then the ethanol was evaporated at 45 °C to form a uniform and transparent liposome film. 1-3 mg of deferroamine mesylate was dissolved in 4-10 mL of phosphate buffer solution (PBS) with pH 7.4. The solution was poured into the formed liposome film and a small amount of glass beads with a diameter of 2-3 mm were added for hydration and washing. The resulting liposome suspension was ultrasonically treated in a water bath and ultrafiltered. The suspension was dialyzed in a dialysis bag with a molecular weight cutoff of 3500-5000 Da to obtain a liposome dispersion.

[0045] (4) Scaffold assembly; Phenyl-2,4,6-trimethylbenzoyl phosphate lithium salt (LAP) was diluted or dissolved in PBS solution to prepare a solution of 0.1-0.5 mg / mL, and methacrylamide gelatin (GelMA) was added. The solution was stirred and dissolved in a water bath at 37°C in the dark. The concentration of methacrylamide gelatin in the solution was 10-30% (w / v %). Liposome dispersion was added to make the final concentration of liposomes 0.1-1 mg / mL to obtain a hydrogel precursor solution. The solid scaffold obtained in step (2) was placed in the liposome dispersion diluted in PBS solution. The concentration of liposomes was 0.5-4 mg / mL. After reaching swelling equilibrium, it was taken out and placed into a mold. The slurry was injected, solidified and demolded to obtain a papillary air cell scaffold. The mass ratio of solid scaffold to hydrogel in the papillary air cell scaffold was approximately 1:(4-6).

[0046] The present invention will now be described in more detail through more specific embodiments.

[0047] Example 1 This embodiment relates to a mastoid air cell stent, the preparation method of which includes the following steps: (1) 3D modeling: Since the mastoid process of SD rats is underdeveloped and almost absent, the cavitary cavity of the auditory bulla is regarded as the mastoid defect. The skull of 8-week-old SD rats is taken and Micro-CT scan (0.5 mm slice thickness) is performed to obtain the three-dimensional structure of the bilateral auditory bulla area. The air cells of different sizes and interconnected are designed according to the morphology of the auditory bulla using 3D MAX software. The three-dimensional image is encoded into a language that can be recognized by the 3D printer and saved for later use.

[0048] (2) Solid scaffold printing: Dilute 0.1 g / mL of 2,2-dimethoxy-2-phenylacetophenone (DMPA) solution with acetone to 5 mg / mL, add F127DA (poloxam bisacrylamide) and PCL (polycaprolactone) at a mass ratio of 1 g / mL, stir for 24 h under light-protected conditions until completely dissolved to obtain 3D printing ink, 3D print the structure model designed and coded in step (1), perform photocrosslinking at a wavelength of 365 nm, soak thoroughly after natural drying, change the water several times to completely remove small molecules, and then place it in a vacuum drying oven at 30°C to dry until the mass basically no longer changes to obtain a solid scaffold, seal and store for later use.

[0049] (3) Liposome preparation: 80 mg egg yolk lecithin (EL), 20 mg cholesterol and 0.2 mg dexamethasone were mixed and dissolved in 5 mL anhydrous ethanol and stirred for 1 h. Then the ethanol was evaporated at 45 °C to form a uniform and transparent liposome film. 2 mg deferroamine mesylate was dissolved in 5 mL phosphate buffer solution (PBS) with pH 7.4. The solution was poured into the formed liposome film and a small amount of glass beads with a diameter of 2-3 mm were added for hydration and washing. The resulting liposome suspension was sonicated in a water bath and filtered through 1 μm, 0.4 μm and 0.2 μm polycarbonate membranes in sequence. Then it was dialyzed in 100 mL PBS solution in the dark with a molecular weight cutoff of 3500 Da. The dialysate was changed every 6 hours and dialyzed for 3 days to obtain the liposome dispersion. It was then sealed and stored in a refrigerator at 4 °C for later use.

[0050] (4) Scaffold assembly; Phenyl-2,4,6-trimethylbenzoyl phosphate lithium salt (LAP) was diluted or dissolved in PBS solution to prepare a solution of 0.25 mg / mL. Methacrylated gelatin (GelMA) was added and stirred and dissolved in a water bath at 37°C in the dark. The concentration of methacrylated gelatin in the solution was 20% (w / v %). Liposome dispersion was added to make the final concentration of liposomes 0.5 mg / mL to obtain a hydrogel precursor solution. The solid scaffold obtained in step (2) was placed in a liposome dispersion diluted in PBS solution with a liposome concentration of 2 mg / mL. After reaching swelling equilibrium, it was taken out and placed in a mold. The hydrogel precursor solution preheated to 37°C was injected into the mold. After waiting for 2 minutes, it was allowed to flow fully and cover the pores between the air cells. Then, it was irradiated with 365 nm ultraviolet light for 2 minutes. After cooling to room temperature, it was carefully demolded to obtain a papillary air cell scaffold.

[0051] Example 2 This embodiment relates to a mastoid air cell stent, the preparation method of which includes the following steps: (1) 3D modeling: Since the mastoid process of SD rats is underdeveloped and almost absent, the cavitary cavity of the auditory bulla is regarded as the mastoid defect. The skull of 8-week-old SD rats is taken and Micro-CT scan (0.5 mm slice thickness) is performed to obtain the three-dimensional structure of the bilateral auditory bulla area. The air cells of different sizes and interconnected are designed according to the morphology of the auditory bulla using 3D MAX software. The three-dimensional image is encoded into a language that can be recognized by the 3D printer and saved for later use.

[0052] (2) Solid scaffold printing: Dilute 0.1 g / mL of 2,2-dimethoxy-2-phenylacetophenone (DMPA) solution with acetone to 4 mg / mL, add F127DA (poloxam bisacrylamide) and PCL (polycaprolactone) at a mass ratio of 1 g / mL according to the material-liquid ratio, stir for 12 h under light-protected conditions until completely dissolved to obtain 3D printing ink, 3D print according to the structure model designed and coded in step (1), perform photocrosslinking at a wavelength of 365 nm, soak thoroughly after natural drying, change water several times to completely remove small molecule substances, and then place it in a vacuum drying oven at 35°C to dry until the mass basically no longer changes to obtain a solid scaffold, seal and store for later use.

[0053] (3) Liposome preparation: 80 mg egg yolk lecithin (EL), 20 mg cholesterol and 0.18 mg dexamethasone were mixed and dissolved in 6 mL of anhydrous ethanol and stirred for 2 h. Then the ethanol was evaporated at 50 °C to form a uniform and transparent liposome film. 1.8 mg deferroamine mesylate was dissolved in 4 mL of phosphate buffer solution (PBS) with pH 7.4. The solution was poured into the formed liposome film and a small amount of glass beads with a diameter of 2-3 mm were added for hydration and washing. The resulting liposome suspension was sonicated in a water bath and filtered through 1 μm, 0.4 μm and 0.2 μm polycarbonate membranes in sequence. Then it was dialyzed in 80 mL of PBS solution in the dark with a molecular weight cutoff of 5000 Da. The dialysate was changed every 5 hours and dialyzed for 3 days to obtain the liposome dispersion. It was then sealed and stored in a refrigerator at 4 °C for later use.

[0054] (4) Scaffold assembly; Phenyl-2,4,6-trimethylbenzoyl phosphate lithium salt (LAP) was diluted or dissolved in PBS solution to prepare a solution of 0.25 mg / mL. Methacrylated gelatin (GelMA) was added and stirred and dissolved in a water bath at 37°C in the dark. The concentration of methacrylated gelatin in the solution was 20% (w / v %). Liposome dispersion was added to make the final concentration of liposomes 0.45 mg / mL, and a hydrogel precursor solution was obtained. The solid scaffold obtained in step (2) was placed in a liposome dispersion diluted in PBS solution. The concentration of liposomes was 1.8 mg / mL. After reaching swelling equilibrium, it was taken out and placed in a mold. The hydrogel precursor solution preheated to 37°C was injected into the mold. After waiting for 2 minutes, it was allowed to flow fully and cover the pores between the air cells. Then, it was irradiated with 365 nm ultraviolet light for 2 minutes. After cooling to room temperature, it was carefully demolded to obtain a papillary air cell scaffold.

[0055] Example 3 This embodiment relates to a mastoid air cell stent, the preparation method of which includes the following steps: (1) 3D modeling: Since the mastoid process of SD rats is underdeveloped and almost absent, the cavitary cavity of the auditory bulla is regarded as the mastoid defect. The skull of 8-week-old SD rats is taken and Micro-CT scan (0.5 mm slice thickness) is performed to obtain the three-dimensional structure of the bilateral auditory bulla area. The air cells of different sizes and interconnected are designed according to the morphology of the auditory bulla using 3D MAX software. The three-dimensional image is encoded into a language that can be recognized by the 3D printer and saved for later use.

[0056] (2) Solid scaffold printing: Dilute 0.1 g / mL of 2,2-dimethoxy-2-phenylacetophenone (DMPA) solution with acetone to 4 mg / mL, add F127DA (poloxam bisacrylamide) and PCL (polycaprolactone) at a mass ratio of 1 g / mL, stir for 24 h under light-protected conditions until completely dissolved to obtain 3D printing ink, 3D print the structure model designed and coded in step (1), perform photocrosslinking at a wavelength of 365 nm, soak thoroughly after natural drying, change the water several times to completely remove small molecules, and then place it in a vacuum drying oven at 35°C to dry until the mass basically no longer changes to obtain a solid scaffold, seal and store for later use.

[0057] (3) Liposome preparation: 80 mg egg yolk lecithin (EL), 20 mg cholesterol and 0.16 mg dexamethasone were mixed and dissolved in 5 mL anhydrous ethanol and stirred for 1 h. Then the ethanol was evaporated at 45 °C to form a uniform and transparent liposome film. 1.6 mg deferroamine mesylate was dissolved in 5 mL phosphate buffer solution (PBS) with pH 7.4. The solution was poured into the formed liposome film and a small amount of glass beads with a diameter of 2-3 mm were added for hydration and washing. The resulting liposome suspension was sonicated in a water bath and filtered through 1 μm, 0.4 μm and 0.2 μm polycarbonate membranes in sequence. Then it was dialyzed in 100 mL PBS solution in the dark with a molecular weight cutoff of 3500 Da. The dialysate was changed every 6 hours and dialyzed for 3 days to obtain a liposome dispersion. The dispersion was sealed and stored in a refrigerator at 4 °C for later use.

[0058] (4) Scaffold assembly; Phenyl-2,4,6-trimethylbenzoyl phosphate lithium salt (LAP) was diluted or dissolved in PBS solution to prepare a solution of 0.25 mg / mL. Methacrylated gelatin (GelMA) was added and stirred and dissolved in a water bath at 37°C in the dark. The concentration of methacrylated gelatin in the solution was 20% (w / v %). Liposome dispersion was added to make the final concentration of liposomes 0.4 mg / mL, and a hydrogel precursor solution was obtained. The solid scaffold obtained in step (2) was placed in a liposome dispersion diluted in PBS solution. The concentration of liposomes was 1.6 mg / mL. After reaching swelling equilibrium, it was taken out and placed in a mold. The hydrogel precursor solution preheated to 37°C was injected into the mold. After waiting for 2 minutes, it was allowed to flow fully and cover the pores between the air cells. Then, it was irradiated with 365 nm ultraviolet light for 2 minutes. After cooling to room temperature, it was carefully demolded to obtain a papillary air cell scaffold.

[0059] Example 4 This embodiment relates to a mastoid air cell stent, the preparation method of which includes the following steps: (1) 3D modeling: Since the mastoid process of SD rats is underdeveloped and almost absent, the cavitary cavity of the auditory bulla is regarded as the mastoid defect. The skull of 8-week-old SD rats is taken and Micro-CT scan (0.5 mm slice thickness) is performed to obtain the three-dimensional structure of the bilateral auditory bulla area. The air cells of different sizes and interconnected are designed according to the morphology of the auditory bulla using 3D MAX software. The three-dimensional image is encoded into a language that can be recognized by the 3D printer and saved for later use.

[0060] (2) Solid scaffold printing: Dilute 0.1 g / mL of 2,2-dimethoxy-2-phenylacetophenone (DMPA) solution with acetone to 5 mg / mL, add F127DA (poloxam bisacrylamide) and PCL (polycaprolactone) at a mass ratio of 1:0.5 at a material-to-liquid ratio of 1 g / mL, stir for 12 h under light-protected conditions until completely dissolved to obtain 3D printing ink, 3D print the structure model designed and coded in step (1), perform photocrosslinking at a wavelength of 365 nm, soak thoroughly after natural drying, change the water several times to completely remove small molecules, and then place it in a vacuum drying oven at 30°C to dry until the mass basically no longer changes to obtain a solid scaffold, seal and store for later use.

[0061] (3) Liposome preparation: 80 mg egg yolk lecithin (EL), 20 mg cholesterol and 0.15 mg dexamethasone were mixed and dissolved in 5 mL anhydrous ethanol and stirred for 1 h. Then the ethanol was evaporated at 45 °C to form a uniform and transparent liposome film. 1.5 mg deferroamine mesylate was dissolved in 5 mL phosphate buffer solution (PBS) with pH 7.4. The solution was poured into the formed liposome film and a small amount of glass beads with a diameter of 2-3 mm were added for hydration and washing. The resulting liposome suspension was sonicated in a water bath and filtered through 1 μm, 0.4 μm and 0.2 μm polycarbonate membranes in sequence. Then it was dialyzed in 100 mL PBS solution in the dark with a molecular weight cutoff of 3500 Da. The dialysate was changed every 6 hours and dialyzed for 3 days to obtain the liposome dispersion. It was then sealed and stored in a refrigerator at 4 °C for later use.

[0062] (4) Scaffold assembly; Phenyl-2,4,6-trimethylbenzoyl phosphate lithium salt (LAP) was diluted or dissolved in PBS solution to prepare a solution of 0.25 mg / mL. Methacrylated gelatin (GelMA) was added and stirred and dissolved in a water bath at 37°C in the dark. The concentration of methacrylated gelatin in the solution was 20% (w / v %). Liposome dispersion was added to make the final concentration of liposomes 0.35 mg / mL, and a hydrogel precursor solution was obtained. The solid scaffold obtained in step (2) was placed in a liposome dispersion diluted in PBS solution with a liposome concentration of 1.5 mg / mL. After reaching swelling equilibrium, it was taken out and placed in a mold. The hydrogel precursor solution preheated to 37°C was injected into the mold. After waiting for 2 minutes, it was allowed to flow fully and cover the pores between the air cells. Then, it was irradiated with 365 nm ultraviolet light for 2 minutes. After cooling to room temperature, it was carefully demolded to obtain a papillary air cell scaffold.

[0063] Example 5 This embodiment relates to a mastoid air cell stent, which differs from Embodiment 1 in that the liposomes do not contain dexamethasone. The preparation method also differs from Embodiment 1 in that step (3) is different. Step (3) in this embodiment is as follows: Liposome preparation: 80 mg egg yolk lecithin (EL) and 20 mg cholesterol were mixed and dissolved in 5 mL of anhydrous ethanol and stirred for 1 h. Then, the ethanol was evaporated at 45 °C to form a uniform and transparent liposome film. 2 mg of deferoxamine mesylate was dissolved in 5 mL of phosphate buffered saline (PBS) at pH 7.4. This solution was poured into the formed liposome film, and a small amount of glass beads with a diameter of 2-3 mm were added for hydration and washing. The resulting liposome suspension was sonicated in a water bath and filtered sequentially through 1 μm, 0.4 μm, and 0.2 μm polycarbonate membranes. Then, it was dialyzed in 100 mL of PBS solution in the dark, with a molecular weight cutoff of 3500 Da. The dialysate was changed every 6 hours and dialyzed for 3 days to obtain a liposome dispersion, which was then sealed and stored in a refrigerator at 4 °C for later use.

[0064] Example 6 This embodiment relates to a mastoid air cell stent, which differs from Embodiment 1 in that the liposomes do not contain deferoxamine mesylate, and its preparation method differs from that of Embodiment 1 in that step (3) is different. Step (3) of this embodiment is as follows: Liposome preparation: 80 mg egg yolk lecithin (EL), 20 mg cholesterol and 0.2 mg dexamethasone were mixed and dissolved in 5 mL anhydrous ethanol and stirred for 1 h. Then, the ethanol was evaporated at 45 °C to form a uniform and transparent liposome film. 5 mL of phosphate buffered saline (PBS) at pH 7.4 was poured into the formed liposome film, and a small amount of glass beads with a diameter of 2-3 mm were added for hydration and washing. The resulting liposome suspension was sonicated in a water bath and filtered sequentially through 1 μm, 0.4 μm and 0.2 μm polycarbonate membranes. Then, it was dialyzed in 100 mL of PBS solution in the dark, with a molecular weight cutoff of 3500 Da. The dialysate was changed every 6 hours and dialyzed for 3 days to obtain a liposome dispersion, which was then sealed and stored in a refrigerator at 4 °C for later use.

[0065] Comparative Example 1 This comparative example relates to a mastoid air cell stent, which differs from Example 1 in that it does not contain liposomes. The preparation method also differs from Example 1 in that step (4) is different. Step (4) of this comparative example is as follows: Scaffold assembly: Lithium phenyl-2,4,6-trimethylbenzoyl phosphate (LAP) was diluted or dissolved in PBS solution to prepare a solution of 0.25 mg / mL. Methacrylated gelatin (GelMA) was added and stirred and dissolved in a water bath at 37°C in the dark. The concentration of methacrylated gelatin in the solution was 20% (w / v %), and a hydrogel precursor solution was obtained. The solid scaffold obtained in step (2) was placed in PBS solution. After swelling equilibrium was reached, it was taken out and placed into a mold. The hydrogel precursor solution preheated to 37°C was injected into the mold. After waiting for 2 minutes, it was allowed to flow fully and cover the pores between the air cells. Then, it was irradiated with 365 nm ultraviolet light for 2 minutes. After cooling to room temperature, it was carefully demolded to obtain a papillary air cell scaffold.

[0066] Experimental Example (1) In vitro drug release performance of liposomes Five mL of each of the following suspensions were placed into dialysis bags with a molecular weight cutoff of 3500: free deferoxamine mesylate (DFO-free), free DEX (DEX-free), liposome-loaded deferoxamine mesylate (DFO-lip), and liposome-loaded dexamethasone (DEX-lip). Each bag was then immersed in 50 mL of PBS solution and subjected to in vitro release curve analysis using a magnetic stirrer at 37°C. A PBS dilution of the same concentration of DFO and DEX was used as a control. At set intervals, 1 mL of PBS was aspirated from the outer dialysis layer, and the concentrations of DFO and DEX were determined by HPLC. Immediately afterward, 1 mL of PBS was added. The release rate (R%) was calculated using the following formula, and in vitro release curves were plotted.

[0067]

[0068] Where Q0 is the total amount of the drug added (mg); C n V represents the concentration of the drug (mg / mL) detected after the nth time the outer layer of PBS is removed; n The volume of the receiving cell is 50 mL; C i V represents the drug concentration (mg / mL) at the i-th point; i This is the volume of each sample taken, i.e., 1 mL.

[0069] Release curves (n=3) of free DFO, free DEX, liposome-loaded DFO, and liposome-loaded DEX in PBS solution at 37°C in vitro. Figure 3 As shown, by Figure 3 It can be seen that liposome encapsulation achieves sustained drug release.

[0070] (2) Evaluation of osteogenic performance Bone marrow mesenchymal stem cells (BMSCs) were digested with trypsin, and the cell suspension concentration was adjusted with conditioned medium before being seeded into 24-well plates (2.0 × 10⁻⁶). 5 (cells / well) lower chamber of Transwell chamber and 6-well plate (10 cells / well) 6 The lower chamber of the Transwell chamber (cells / well). 24-well plates were used for staining, and 6-well plates were used for RNA or protein extraction. When adhesion and confluence reached 80%, the cells were cultured under hypoxia for 12 hours (37°C, 1% O2, 5% CO2), then grouped according to the examples and cultured under normoxic conditions. The Transwell chambers were removed at the predetermined time point (21 days). ALP and Alizarin Red staining were used to evaluate the in vitro osteogenic effect.

[0071] (3) Evaluation of in vitro angiogenesis performance Rabbit umbilical vein endothelial cells (UVECs) were digested with trypsin, and the cell suspension concentration was adjusted with conditioned medium before being seeded into the lower chamber of a 6-well Transwell plate. When 60% adherence and confluence were achieved, the cells were cultured under hypoxia for 12 hours (37°C, 1% O2, 5% CO2), and then grouped according to different implementation examples. A portion of the 6-well plates was used for matrix gelation experiments.

[0072] (4) Evaluation of in vitro inflammation suppression After gentle pipetting, RAW264.7 cells were adjusted to conditioned medium concentration and seeded into 6-well plates (10⁶ cells / wells). 6The lower chamber of the Transwell chamber (cells / well) was used. Once 80% adhesion and confluence were achieved, the cells were processed according to the example grouping. After 12 hours of treatment, the Transwell chamber was removed, and qPCR was used to verify inflammatory markers, including inducible nitric oxide synthase (iNOS) and tumor necrosis factor-α (TNF-α).

[0073] Using the osteogenic test group without the addition of the examples or comparative examples as a blank control, the evaluation parameters of the osteogenic, angiogenic, and anti-inflammatory properties of the mastoid air cell scaffolds prepared in each example and comparative example are shown in Table 1. The results in Table 1 show that the liposome-loaded 3D-printed scaffold prepared in the embodiments of the present invention can significantly inhibit inflammation, promote angiogenesis, and promote osteogenic formation. In addition to inhibiting inflammation, dexamethasone can also synergistically promote normal angiogenesis and bone formation with deferoxamine mesylate. It is expected that the solid scaffold 3D biomimetic mastoid air cell structure, as the mechanical support of the scaffold, will gradually degrade or physically dissolve to form air cells in the later stages of new bone formation.

[0074] Table 1. Evaluation parameters of osteogenic, angiogenic, and anti-inflammatory properties of the mastoid air cell stents prepared in each embodiment and comparative example.

[0075] The degradation times of the solid scaffold and hydrogel in the mastoid air cell scaffolds prepared in Examples 1-4 are shown in Table 2. According to Table 2, the hydrogel in the examples has a shorter degradation time, approximately 28 days. After degradation, the soft material can form a bone wall, and after degradation, the hard material can form air cells. By adjusting the ratio of F127DA and PCL, the degradation time of the solid scaffold can be controlled from 80 days to 200 days.

[0076] Table 2. Degradation time of solid scaffolds and hydrogels in papillary air cell stents of Examples 1-4

[0077] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A mastoid air cell stent, characterized in that, Includes a biodegradable solid scaffold having air cell pores; It also includes biodegradable hydrogels containing liposomes loaded with drugs, the drugs including one or a combination of angiogenic drugs, osteoproliferative drugs, and anti-inflammatory drugs. The hydrogel at least partially fills the pores of the air chamber and / or at least partially covers the surface of the solid scaffold.

2. The mastoid air cell stent according to claim 1, characterized in that, The solid scaffold material comprises a polymer of alkenyl poloxamer and polycaprolactone; and / or, the hydrogel material comprises a polymer of at least one of alkenyl-modified gelatin, alkenyl-modified hyaluronic acid, and alkenyl-modified sodium alginate.

3. A mastoid air cell stent according to claim 2, characterized in that, The mass ratio of the polymerized alkenyl poloxamer to the polycaprolactone is 1:(0.1-5).

4. A mastoid air cell stent according to claim 1, characterized in that, The mass ratio of the solid scaffold to the hydrogel is 1:(1-10); and / or, the concentration of the liposomes in the hydrogel is 0.1-1.0 mg / mL.

5. A mastoid air cell stent according to claim 1, characterized in that, The drugs include deferoxamine mesylate and dexamethasone.

6. A method for preparing a mastoid air cell stent according to any one of claims 1-5, characterized in that, Includes the following steps: (1) Prepare a solid scaffold with air cell pores according to the pore structure of the mastoid air cells; (2) Preparation of drug-loaded liposomes; (3) The solid scaffold is placed in a solution containing the liposomes for swelling equilibrium; (4) The hydrogel precursor solution containing liposomes is placed in a mold containing a solid scaffold after swelling equilibrium and solidified to obtain the papillary air cell scaffold.

7. The preparation method according to claim 6, characterized in that, The solid scaffold is fabricated using a 3D printing method.

8. The preparation method according to claim 6, characterized in that, The hydrogel precursor solution includes a crosslinking monomer at a concentration of 10-30 g / 100 mL; and / or, the concentration of the liposomes in the hydrogel precursor solution is 0.1-1 mg / mL.

9. The preparation method according to claim 6, characterized in that, The liposomes in step (2) are prepared by thin-film rotary evaporation-ultrasound method.

10. The use of the mastoid air cell stent according to any one of claims 1-5 in promoting angiogenesis or bone repair.

Images