A regenerative bone scaffold capable of sequentially releasing drugs, and its preparation method and application

Abstract

The present invention belongs to the field of regenerative medicine technology, and specifically relates to a preparation method and application of a regenerative bone scaffold capable of sequentially releasing drugs. 2+ and drugs (such as dexamethasone) were loaded in different forms, making the Cu 2+ It can be released early and quickly as the stent degrades and promote the formation of new blood vessels; compared with Cu 2+ The special mesoporous structure of mesoporous silica microspheres (MSNs) can make the loaded drugs be released slowly and long-term, thus promoting the later stage of osteogenesis. 2+ The sequential release of thiazolidinone and dexamethasone also enables the scaffold to better achieve the coupling of angiogenesis and osteogenesis in the process of repairing bone defects, simulating the defect repair process of natural bone in the human body.

Description

Technical Field

[0001] The present invention belongs to the technical field of regenerative medicine, and in particular relates to a regenerative bone scaffold capable of sequentially releasing drugs, and a preparation method and application thereof. Background Art

[0002] Trauma, infection, osteoporosis, obesity, congenital bone defects, and tumor resection are factors that can easily cause large bone defects, which seriously affect the patient's normal life. It is well known that the human body cannot completely heal large bone defects. At the same time, the process of bone damage is often accompanied by traumatic peripheral vascular damage and infection at the site of damaged tissue, which further increases the difficulty of repairing large bone defects. Therefore, the repair of large bone defects remains a medical problem that plagues clinical practice, and people also have a great demand for the repair of large bone defects.

[0003] At present, in clinical practice, defective bone tissue is generally repaired by implanting a graft. Traditional treatments for common bone defect repair include autologous bone transplantation, allogeneic bone transplantation, artificial bone transplantation, and artificial bone replacement, but these methods have problems such as insufficient donor sources, immune rejection, secondary damage, and poor biocompatibility. In particular, it is extremely difficult to repair traumatic peripheral vascular damage and defective bone tissue under the premise of avoiding infection at the site of bone injury. In recent years, the emergence of bone tissue engineering technology may make up for the shortcomings of traditional bone defect repair methods. This technology can prepare bone scaffolds with good biocompatibility, biodegradability, promotion of angiogenesis in defective bone tissue, and anti-infection, making this technology a research hotspot for relevant scientific researchers.

[0004] Bone tissue is a highly vascularized human tissue, and natural bone development and defect regeneration are complex and highly coordinated processes. In recent years, vascularization of tissue-engineered bone has become a key factor in repairing large bone tissue defects, and angiogenesis is considered a prerequisite for bone formation. Studies have shown that angiogenesis and bone regeneration are a highly coupled process, including the early formation of new blood vessels and accompanying bone regeneration, and this process requires the provision of sufficient oxygen and nutrients. Therefore, the preparation of a regenerative bone scaffold that can mimic the temporal coupling of angiogenesis and osteogenesis and the natural bone healing process remains a major challenge in the field of bone tissue regeneration. Summary of the Invention

[0005] To address the challenges of the prior art, the present invention provides a sequentially drug-releasing regenerative bone scaffold, its preparation method, and its application. The regenerative bone scaffold produced using the present invention's preparation method effectively couples angiogenesis and osteogenesis during bone defect repair, mimicking the body's natural bone defect repair process.

[0006] In order to achieve the above objectives, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing a bone regeneration scaffold capable of sequentially releasing drugs, comprising the following steps:

[0008] Providing drug-loaded mesoporous silica microspheres; the drug includes dexamethasone;

[0009] mixing methacrylic anhydride gelatin, sodium alginate, a photoinitiator, hydroxyapatite, an inorganic copper salt and the drug-loaded mesoporous silica microspheres to obtain a bio-ink;

[0010] The aqueous solution of the sacrificial bio-ink material is used as the core layer material, and the bio-ink is used as the shell layer material. The aqueous solution of the sacrificial bio-ink material and the bio-ink are coaxially extruded and printed and photocrosslinked to obtain a pre-crosslinked regenerative bone scaffold; the temperatures of the coaxial extrusion printing and photocrosslinking are independently 3 to 5°C;

[0011] mixing the pre-crosslinked regenerative bone scaffold and a calcium salt solution, and performing re-crosslinking to obtain a primary regenerative bone scaffold;

[0012] The sacrificial bio-ink material is removed from the primary regenerated bone scaffold, and the obtained bone scaffold with a hollow structure is sequentially frozen and freeze-dried to obtain the regenerated bone scaffold.

[0013] Preferably, the method for preparing the drug-loaded mesoporous silica microspheres comprises the following steps:

[0014] The mesoporous silica microspheres and the drug solution are mixed, the obtained suspension is vacuum loaded, and then solid-liquid separation is performed to obtain the drug-loaded mesoporous silica microspheres.

[0015] Preferably, the mass ratio of the drug to the mesoporous silica microspheres is 1:300-400.

[0016] Preferably, the vacuum degree of the vacuum load is 8-10 kPa, the temperature is 20-30° C., and the time is 4-8 hours.

[0017] Preferably, the diameter of the mesoporous silica microspheres is 500-900 nm, and the specific surface area is 300-600 m 2 / g.

[0018] Preferably, the photoinitiator includes one or more of lithium phenyl-2,4,6-trimethylbenzoylphosphite, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, 2,2'-azo(2-methyl-N-(2-hydroxyethyl)propionamide) and ethyl 2,4,6-trimethylbenzoylphenylphosphonate.

[0019] Preferably, the photocrosslinking is ultraviolet light crosslinking, the wavelength of the ultraviolet light is 400-410 nm, and the photocrosslinking time is 2-4 minutes.

[0020] Preferably, the concentration of the calcium salt solution is 8-12 wt %, the temperature of the re-crosslinking is 3-5° C., and the time is 25-35 min.

[0021] The present invention also provides a regenerated bone scaffold prepared by the preparation method. The pore size between fibers of the composite bone scaffold is 400-600 μm, and the porosity is 70-90%.

[0022] The present invention also provides the use of the above-mentioned regenerative bone scaffold in preparing a material for treating bone defects and repairing them.

[0023] The present invention provides a preparation method of a regenerative bone scaffold capable of sequentially releasing drugs, comprising the following steps: providing drug-loaded mesoporous silica microspheres; the drug comprising dexamethasone; mixing methacrylic anhydride-modified gelatin, sodium alginate, a photoinitiator, hydroxyapatite, an inorganic copper salt, and the drug-loaded mesoporous silica microspheres to obtain bio-ink; using an aqueous solution of a sacrificial bio-ink material as a core layer material and the bio-ink as a shell layer material, coaxially extrusion-printing and photo-crosslinking the aqueous solution of the sacrificial bio-ink material and the bio-ink to obtain a pre-crosslinked regenerative bone scaffold; the temperatures of the coaxial extrusion printing and photo-crosslinking are independently 3-5°C; mixing the pre-crosslinked regenerative bone scaffold with a calcium salt solution, and re-crosslinking to obtain a primary regenerative bone scaffold; removing the sacrificial bio-ink material from the primary regenerative bone scaffold, and sequentially freezing and freeze-drying the obtained bone scaffold having a hollow structure to obtain the regenerative bone scaffold.

[0024] The regenerative bone scaffold for sequential drug release provided by the present invention is made of non-toxic materials with good biodegradability and biocompatibility, which facilitates the attachment and proliferation of cells in the defective part of human bone tissue. 2+ It can promote angiogenesis and the growth and mineralization of bone tissue, has a certain antibacterial effect, and can be combined with SA to Cu 2 + -SA cross-linked network. 2+ and drugs (dexamethasone) were loaded in different forms, making the Cu 2+ It can be released early and quickly as the stent degrades and promote the formation of new blood vessels; compared with Cu 2+ The special mesoporous structure of mesoporous silica microspheres (MSNs) can make the loaded drugs be released slowly and long-term, thus promoting the later stage of osteogenesis. 2+The sequential release of Cu and dexamethasone also enables the scaffold to better achieve the coupling of angiogenesis and osteogenesis in the process of repairing bone defects, simulating the defect repair process of natural bone in the human body. The common sequential release drug combination in regenerative bone scaffolds, namely bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF), has high cost, short half-life, and excessive side effects; in comparison, the Cu used in the present invention 2+ and DEX can better achieve the coupling of angiogenesis and osteogenesis while making up for the deficiencies of BMP-2 and VEGF. 2+ -SA, Ca 2+ Compared to single cross-linked networks, cross-linked networks such as -SA and GelMA-photoinitiator can further improve the mechanical properties of the scaffold. Furthermore, a composite process combining coaxial 3D printing, sacrificial material processing, and freeze-drying technology has been used to prepare bone scaffolds with hollow channels and fine macro- and micro-pore sizes. This facilitates material exchange between the surrounding tissues and cells, promotes the migration and expansion of surrounding cells, and is more conducive to the repair of defective bone tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The figure is a flow chart for the preparation of drug-loaded mesoporous silica microspheres;

[0026] Figure 2 Flow chart for the preparation of primary regenerative bone scaffolds;

[0027] Figure 3 A flow chart for the preparation of a regenerative bone scaffold with hollow channels and a schematic diagram of its internal structure;

[0028] Figure 4 Schematic diagram of the mechanism by which regenerative bone scaffolds promote angiogenesis and osteogenic differentiation. DETAILED DESCRIPTION

[0029] The present invention provides a method for preparing a bone regeneration scaffold capable of sequentially releasing drugs, comprising the following steps:

[0030] Providing drug-loaded mesoporous silica microspheres; the drug includes dexamethasone;

[0031] mixing methacrylic anhydride gelatin, sodium alginate, a photoinitiator, hydroxyapatite, an inorganic copper salt and the drug-loaded mesoporous silica microspheres to obtain a bio-ink;

[0032] The aqueous solution of the sacrificial bio-ink material is used as the core layer material, and the bio-ink is used as the shell layer material. The aqueous solution of the sacrificial bio-ink material and the bio-ink are coaxially extruded and printed and photocrosslinked to obtain a pre-crosslinked regenerative bone scaffold; the temperatures of the coaxial extrusion printing and photocrosslinking are independently 3 to 5°C;

[0033] mixing the pre-crosslinked regenerative bone scaffold and a calcium salt solution, and performing re-crosslinking to obtain a primary regenerative bone scaffold;

[0034] The sacrificial bio-ink material is removed from the primary regenerated bone scaffold, and the obtained bone scaffold with a hollow structure is sequentially frozen and freeze-dried to obtain the regenerated bone scaffold.

[0035] In the present invention, unless otherwise specified, the raw materials used in the present invention are preferably commercially available products.

[0036] The present invention provides drug-loaded mesoporous silica microspheres.

[0037] In the present invention, the drug-loaded mesoporous silica microspheres are preferably prepared by the following steps:

[0038] The mesoporous silica microspheres and the drug solution are mixed, the obtained suspension is vacuum loaded, and then solid-liquid separation is performed to obtain the drug-loaded mesoporous silica microspheres.

[0039] In the present invention, the mixing method is preferably ultrasonic dispersion, the frequency of the ultrasonic dispersion is preferably 35 to 45 kHz, and the time is preferably 15 minutes.

[0040] In the present invention, the vacuum degree of the vacuum load is preferably 8-10 kPa, the temperature is preferably 20-30° C., and the time is preferably 4-8 hours. In the present invention, the solid-liquid separation method is preferably centrifugation, and the centrifugal speed is preferably 1800-2200 rpm, more preferably 2000 rpm.

[0041] In the present invention, the drug solution preferably comprises a drug and phosphate buffered saline; the drug preferably comprises dexamethasone. In the present invention, the concentration of the drug solution is preferably 0.15 to 0.25 mg / mL, more preferably 0.2 mg / mL.

[0042] The drug dexamethasone (DEX) used in the present invention is a synthetic glucocorticoid. DEX can stimulate osteoblast differentiation and bone tissue formation in vivo, has high stability and osteogenic activity, and has certain antibacterial activity.

[0043] In the present invention, the diameter of the mesoporous silica microspheres is preferably 500 to 900 nm, more preferably 600 to 800 nm; the specific surface area of ​​the mesoporous silica microspheres is preferably 300 to 600 m 2 / g, more preferably 500m 2 In the present invention, the mass ratio of the mesoporous silica microspheres to the drug is preferably 300-400:1, more preferably 350:1.

[0044] In the present invention, the mixing is preferably ultrasonic mixing and stirring mixing performed in sequence, the frequency of the ultrasonic mixing is preferably 35-45kHz, more preferably 40kHz, and the time is preferably 10-20min, more preferably 15min; the rotation speed of the stirring mixing is preferably 400-600rpm, more preferably 500rpm, and the time is preferably 20-30h, more preferably 25h.

[0045] In the present invention, the vacuum degree of the vacuum load is preferably 8-10 kPa, the time is preferably 4-8 hours, more preferably 5-6 hours. In the present invention, the solid-liquid separation method is preferably centrifugation, the speed of the centrifugation is preferably 1800-2200 rpm, and the time is preferably 4-6 minutes.

[0046] In the present invention, after the solid-liquid separation, the solid phase obtained by the solid-liquid separation is preferably washed and dried. The washing reagent is preferably phosphate buffered saline, and the number of washings is preferably 3. In the present invention, the drying is preferably freeze-drying, and the freeze-drying temperature is preferably -75 to -80°C, and the time is preferably 10 to 15 hours, more preferably 12 hours.

[0047] Figure 1 The preparation flow chart of drug-loaded mesoporous silica microspheres is shown in Figure 2. Taking the drug DEX as an example, Figure 1 It can be seen that: DEX was added to 10 mL of phosphate buffered saline (PBS solution) and stirred evenly, and the obtained DEX solution and mesoporous silica microspheres were ultrasonically mixed, stirred, centrifuged, washed and vacuum freeze-dried in sequence to obtain drug-loaded mesoporous silica microspheres, namely DEX@MSNs.

[0048] After obtaining drug-loaded mesoporous silica microspheres, the present invention mixes methacrylic anhydride gelatin, sodium alginate, a photoinitiator, hydroxyapatite, an inorganic copper salt and the drug-loaded mesoporous silica microspheres to obtain biological ink.

[0049] In the present invention, the photoinitiator preferably includes one or more of phenyl-2,4,6-trimethylbenzoyl lithium phosphite, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, 2,2'-azo(2-methyl-N-(2-hydroxyethyl)propionamide) and ethyl 2,4,6-trimethylbenzoylphenylphosphonate, more preferably phenyl-2,4,6-trimethylbenzoyl lithium phosphite.

[0050] In the present invention, the inorganic copper salt preferably includes one or more of CuCl2, CuSO4, Cu(NO3)2 and Cu(CH3COO)2, more preferably CuCl2.

[0051] In the present invention, the mass ratio of the methacrylic anhydride gelatin to sodium alginate is preferably 4-6:3-5, more preferably 5:4. In the present invention, the mass ratio of the methacrylic anhydride gelatin to the photoinitiator is preferably 1:0.04-0.24, more preferably 1:0.1-0.2. In the present invention, the mass ratio of the methacrylic anhydride gelatin to hydroxyapatite is preferably 0.4-0.6:0.15-0.25, more preferably 0.5:0.15-0.25. In the present invention, the mass ratio of the methacrylic anhydride gelatin to the inorganic copper salt is preferably 400-600:1-2, more preferably 500:1-2. In the present invention, the mass ratio of the methacrylic anhydride gelatin to the drug-loaded mesoporous silica microspheres is preferably 0.4-0.6:0.08-0.12, more preferably 0.5:0.08-0.12.

[0052] After obtaining the bio-ink, the present invention uses an aqueous solution of a sacrificial bio-ink material as a core layer material and the bio-ink as a shell layer material, and coaxially extrudes and prints the aqueous solution of the sacrificial bio-ink material and the bio-ink and photocrosslinks them to obtain a pre-crosslinked regenerative bone scaffold.

[0053] In the present invention, the preparation of the aqueous solution of the sacrificial bio-ink material preferably includes: stirring and mixing the sacrificial bio-ink material and water to obtain the aqueous solution of the sacrificial bio-ink material.

[0054] In the present invention, the sacrificial bio-ink material preferably includes one or more of gelatin, polyoxyethylene polyoxypropylene ether, and polyethylene glycol, with gelatin being more preferred. In the present invention, the mass ratio of the sacrificial bio-ink material to water is preferably 0.4-0.6:8-12, more preferably 0.5:8-12. In the present invention, the stirring and mixing is preferably performed at a speed of 200 rpm and for a time of 0.5-1.5 hours, more preferably 1 hour.

[0055] In the present invention, the temperatures for the coaxial extrusion printing and photocrosslinking are independently 3-5°C, preferably 4°C. In the present invention, the photocrosslinking time is preferably 3-4 minutes. In the present invention, the coaxial extrusion printing conditions include: the shell needle inner diameter of the coaxial printing needle is preferably 0.7-0.8 mm, and the core needle inner diameter is preferably 0.2-0.3 mm. In the present invention, photocrosslinking is performed simultaneously with the coaxial extrusion.

[0056] In an embodiment of the present invention, the coaxial extrusion printing is preferably printed in a "well" grid shape, with adjacent fibers not connected to each other. The coaxial extrusion printing preferably has a single layer height of 0.75 to 0.85 mm.

[0057] After obtaining the pre-crosslinked regenerative bone scaffold, the present invention mixes the pre-crosslinked regenerative bone scaffold with a calcium salt solution and performs re-crosslinking to obtain a primary regenerative bone scaffold.

[0058] In the present invention, the calcium salt solution preferably includes a CaCl2 solution, and the concentration of the calcium salt solution is preferably 8 to 12 wt %, more preferably 10 wt %. In the present invention, the mass ratio of the pre-crosslinked regenerative bone scaffold to the CaCl2 solution is preferably 1:0.4 to 0.6, more preferably 1:0.5.

[0059] In the present invention, the re-crosslinking temperature is preferably 3-5°C, more preferably 4°C, and the re-crosslinking time is preferably 25-35 min, more preferably 30 min.

[0060] Figure 2 The flow chart for the preparation of primary regenerative bone scaffolds is as follows: Figure 2 It can be seen that: methacrylic anhydride gelatin (GelMA), sodium alginate (SA), photoinitiator (LAP), hydroxyapatite (HAP), inorganic copper salt (Cu 2+ ) and drug-loaded mesoporous silica microspheres (DEX@MSNs) to obtain bio-ink; using an aqueous solution of a sacrificial bio-ink material as a core layer material and the bio-ink as a shell layer material, the aqueous solution of the sacrificial bio-ink material and the bio-ink are coaxially extruded and printed and photocrosslinked to obtain a pre-crosslinked regenerative bone scaffold; the temperatures of the coaxial extrusion and photocrosslinking are independently 3 to 5°C; the pre-crosslinked regenerative bone scaffold is mixed with a calcium salt solution and re-crosslinked to obtain a primary regenerative bone scaffold.

[0061] After obtaining the primary regenerated bone scaffold, the present invention removes the sacrificial bio-ink material from the primary regenerated bone scaffold, and the obtained bone scaffold with hollow channels is sequentially frozen and freeze-dried to obtain the regenerated bone scaffold.

[0062] In the present invention, the method for removing the sacrificial bio-ink material comprises the following steps:

[0063] The sacrificial bio-ink material in the primary regenerative bone scaffold is placed in an environment of 35 to 40° C. for 30 to 50 minutes, and the sacrificial bio-ink material is dissolved by utilizing its temperature-sensitive properties to obtain a bone scaffold with hollow channels.

[0064] Figure 3 The flow chart of the preparation of regenerative bone scaffold with hollow channels and the schematic diagram of the internal structure composition are shown in Figure 2. Figure 3 It can be seen that the sacrificial bio-ink material (core layer) is removed from the primary regenerated bone scaffold and dissolved, and the obtained bone scaffold with hollow channels is sequentially frozen and freeze-dried to obtain the regenerated bone scaffold.

[0065] In the present invention, before freezing, the bone scaffold with hollow channels is preferably washed, and the washing agent is preferably phosphate buffered saline (PBS solution). In the present invention, the freezing temperature is preferably -75 to -80°C, more preferably -78°C, and the time is preferably 6 to 10 hours, more preferably 7 to 8 hours. In the present invention, the drying is preferably vacuum freeze drying, and the conditions of the vacuum freeze drying preferably include: the vacuum degree is preferably 8 to 10 kPa, the temperature is preferably -75 to -80°C, and the time is preferably 6 to 10 hours, more preferably 7 to 8 hours.

[0066] The present invention also provides a regenerated bone scaffold prepared by the above preparation method. The pore size between fibers of the composite bone scaffold is preferably 400-600 μm, more preferably 500 μm; the porosity of the composite bone scaffold is preferably 70-90%, more preferably 80%.

[0067] The present invention also provides the use of the above-mentioned regenerated bone scaffold in preparing a material for treating bone defects and repairing the bone.

[0068] Figure 4 Schematic diagram of the mechanism of promoting angiogenesis and osteogenic differentiation by regenerating bone scaffolds, such as Figure 4 As shown in Figure 2, DEX can stimulate osteoblast differentiation and bone tissue formation in vivo, has high stability and osteogenic activity, and has certain antibacterial activity; in addition, the Cu in the scaffold 2+ It can promote angiogenesis and the growth and mineralization of bone tissue, and also has a certain antibacterial effect, and can be combined with SA to Cu 2+ -SA exists in the form of a cross-linked network.

[0069] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0070] Example 1

[0071] (1) Preparation of DEX@MSNs powder

[0072] First, 2 mg of DEX was added to 10 mL of phosphate-buffered saline and stirred evenly to form a DEX solution with a concentration of 0.2 mg / mL.

[0073] 0.2 g of MSNs was added to 10 mL of DEX solution to form a mixed solution of MSNs and DEX. The mixed solution was ultrasonically dispersed at a frequency of 40 kHz for 15 min and stirred at room temperature for 24 h to form a DEX@MSNs suspension.

[0074] After the DEX@MSNs suspension was placed under vacuum for 6 hours, the suspension was centrifuged at a speed of 2000 rpm. The precipitate obtained by centrifugation was washed three times with PBS solution, and the supernatant was removed to collect the precipitate DEX@MSNs. The collected precipitate DEX@MSNs was freeze-dried at -78°C for 12 hours to obtain dry DEX@MSNs powder.

[0075] By performing an absorbance test on the DEX@MSNs supernatant prepared in Example 1 and comparing it with the DEX concentration-absorbance standard curve, it can be calculated that the drug loading capacity of the prepared DEX@MSNs is 3.2 μg of DEX per 1 mg of MSNs.

[0076] (2) Preparation of bio-ink

[0077] At 37°C, 0.5 g of GelMA, 0.025 g of LAP, 0.4 g of SA, 0.2 g of HAP, 1.35 mg of anhydrous CuCl2 particles, and 0.1 g of DEX@MSNs powder were added to 10 mL of deionized water and stirred for 2 h to obtain the bio-ink.

[0078] Aqueous solution of sacrificial bio-ink material: 0.5 g of Gel was dissolved in 10 mL of deionized water at 37° C. and stirred for 1 h to prepare a Gel solution.

[0079] (3) Pre-crosslinked regenerative bone scaffold

[0080] The aqueous solution of bio-ink and sacrificial bio-ink material is coaxially extruded and printed through a coaxial printing needle, wherein the aqueous solution of the sacrificial bio-ink material is the core layer material and the bio-ink is the shell layer material. The inner diameter of the shell layer needle in the coaxial printing needle is 0.72 mm, and the inner diameter of the core layer needle is 0.26 mm.

[0081] During the printing process, the bio-ink was photocross-linked (cross-linked while extruding) using ultraviolet light with a wavelength of 405nm to form a GelMA-LAP photocross-linked network structure, which was further printed to form a "well"-shaped pre-cross-linked regenerative bone scaffold with dimensions of 15mm (length) × 15mm (width) × 6mm (height).

[0082] The pre-crosslinked regenerative bone scaffold was mixed with 0.5 mL of 10 wt % CaCl 2 solution and re-crosslinked for 30 min (temperature was 4° C.) to obtain a primary regenerative bone scaffold.

[0083] (4) Regenerative bone scaffold

[0084] The primary regenerated bone scaffold was placed in an environment of 37°C for 40 minutes, and the Gel was dissolved and flowed out of the scaffold using its thermosensitive properties, thereby forming a regenerated bone scaffold with hollow channels.

[0085] Then, the scaffold was washed three times with deionized water to remove uncrosslinked CaCl2 and residual Gel, and then the scaffold was placed in a -80°C refrigerator and frozen for 8 hours. Finally, the frozen scaffold was vacuum freeze-dried (temperature of -78°C, vacuum degree of 9 kPa) for 8 hours to obtain the regenerated bone scaffold.

[0086] Comparative Example 1

[0087] The only difference from Example 1 is that “0.1 g DEX@MSNs powder” is replaced by “0.64 mg DEX”.

[0088] Comparative Example 2

[0089] (1) Preparation of DEX@MSNs powder

[0090] First, 2 mg of DEX was added to 10 mL of phosphate-buffered saline and stirred evenly to form a DEX solution with a concentration of 0.2 mg / mL.

[0091] 0.2 g of MSNs was added to 10 mL of DEX solution to form a mixed solution of MSNs and DEX. The mixed solution was ultrasonically dispersed at a frequency of 40 kHz for 15 min and stirred at room temperature for 24 h to form a DEX@MSNs suspension.

[0092] After the DEX@MSNs suspension was placed under vacuum for 6 hours, the suspension was centrifuged at 2000 rpm and washed three times with PBS. The supernatant was removed and the precipitate DEX@MSNs was collected. The collected precipitate DEX@MSNs was freeze-dried at -78°C for 12 hours to obtain dry DEX@MSNs powder.

[0093] By performing an absorbance test on the supernatant of DEX@MSNs prepared in Example 1 and comparing it with the DEX standard curve, it can be calculated that the drug loading capacity of the prepared DEX@MSNs is 3.2 μg of DEX per 1 mg of MSNs.

[0094] (2) Preparation of bio-ink

[0095] At 37°C, 0.5 g of GelMA, 0.025 g of LAP, 0.4 g of SA, 0.2 g of HAP, 1.35 mg of anhydrous CuCl2 particles, and 0.1 g of DEX@MSNs powder were added to 10 mL of deionized water and stirred for 2 h to obtain the bio-ink.

[0096] Aqueous solution of sacrificial bio-ink material: 0.5 g of Gel was dissolved in 10 mL of deionized water at 37° C. and stirred for 1 h to prepare a Gel solution.

[0097] (3) Pre-crosslinked regenerative bone scaffold

[0098] The bio-ink was extruded and printed and photo-crosslinked using a uniaxial printing needle, and the bio-ink was photo-crosslinked using ultraviolet light with a wavelength of 405 nm (cross-linking while extruding and printing) to form a GelMA-LAP photo-crosslinked network structure, which was further printed to form a "well"-shaped pre-cross-linked regenerative bone scaffold with dimensions of 15 mm (length) × 15 mm (width) × 6 mm (height).

[0099] The pre-crosslinked regenerative bone scaffold was mixed with 0.5 mL of 10 wt % CaCl 2 solution and re-crosslinked for 30 min (temperature was 4° C.) to obtain a primary regenerative bone scaffold.

[0100] (4) Regenerative bone scaffold

[0101] The primary regenerated bone scaffold was placed in an environment of 37°C for 40 minutes, and the Gel was dissolved and flowed out of the scaffold using its thermosensitive properties, thereby forming a regenerated bone scaffold with hollow channels.

[0102] Then, the scaffold was washed three times with deionized water to remove uncrosslinked CaCl2 and residual Gel, and then the scaffold was placed in a -80°C refrigerator and frozen for 8 hours. Finally, the frozen scaffold was vacuum freeze-dried (temperature of -78°C, vacuum degree of 9 kPa) for 8 hours to obtain the regenerated bone scaffold.

[0103] The present invention tested the regenerated bone scaffolds prepared in Example 1 and Comparative Examples 1-2, and the determination method was as follows:

[0104] (1) The compressive strength of the three brackets was tested using a universal mechanical testing machine, and the compression modulus was calculated;

[0105] (2) CCK-8 assay of bone marrow mesenchymal stem cells was performed on the three scaffolds, and the cell proliferation rate on day 3 compared with day 1 was calculated;

[0106] (3) Conduct drug release experiments on the three stents for 21 days and calculate Cu 2+ and drug release rate of DEX;

[0107] (4) The skull of rats was modeled and three types of scaffolds were implanted into the model. After 6 weeks, the ratio of the volume of new blood vessels and new bone volume in the scaffold to the total volume of the scaffold was analyzed and calculated.

[0108] The above experimental test results are shown in Table 1 (the stents prepared in Example 1, Comparative Example 1 and Comparative Example 2 are respectively denoted as Stent 1, Stent 2 and Stent 3).

[0109] Table 1 Test results of Example 1, Comparative Example 1 and Comparative Example 2

[0110] Example 1 (Stand 1) Comparative Example 1 (Stand 2) Comparative Example 2 (Stand 3) Compression modulus (MPa) 8.5 6.2 12.7 Cell proliferation rate (%) 90.6 88.3 80.1 <![CDATA[Cu 2+ Drug release rate (%)]]> 95.2 94.5 92.9 DEX drug release rate (%) 55.9 90.6 50.3 Neovascularization volume ratio (%) 12.6 10.1 8.2 New bone tissue volume percentage (%) 25.8 16.4 18.3

[0111] The following conclusions can be drawn from Table 1:

[0112] (1) Compressive strength: Compared with the solid stent 3, the compression performance of stents 1 and 2 is reduced to a certain extent due to their hollow channels. In addition, the compressive performance of stent 1 is improved compared with that of stent 2, possibly because stent 1 contains drug-loaded microspheres.

[0113] (2) Cell proliferation rate: Compared with the solid scaffold 3, the cell proliferation rates of scaffolds 1 and 2 are close to 90%, which may be due to the hollow channels in scaffolds 1 and 2. This special structure can promote cell adhesion and proliferation.

[0114] (3)Cu 2+ and DEX drug release rate: From the test results, it can be seen that when the drug is released for 21 days, the Cu 2+ The release of DEX was basically complete. For DEX, after 21 days of drug release, except for the DEX of scaffold 2 which was basically completely released, the DEX release of scaffolds 1 and 3 was only about 50%. This may be due to the presence of drug-loaded microspheres DEX@MSNs, which enabled the DEX loaded in the microspheres to be slowly released, thereby better promoting new bone formation.

[0115] (4) Proportion of volume of new blood vessels: Compared with solid stent 3, the volume of new blood vessels in stents 1 and 2 accounts for more than 10%. This may be because the presence of hollow channels in stents 1 and 2 makes it easier for blood vessels sprouting from the adjacent defective bone tissue to enter these two stents to form new blood vessel tissue.

[0116] (5) New bone tissue volume ratio: From the results, it can be seen that scaffold 1 has a higher new bone tissue volume ratio, which may be due to the sequential release of Cu by scaffold 1. 2+ and DEX, Cu 2+ The faster release of DEX and the slower release of blood vessels followed by osteogenesis are more consistent with the body's natural bone defect repair process. Secondly, the presence of hollow channels also promotes cell migration, proliferation, and material exchange, significantly accelerating the repair process of defective bone tissue.

[0117] The above results show that compared with Comparative Examples 1 and 2, Example 1 can release Cu 2+ and DEX, and has a special hollow channel structure, which can better repair large bone tissue defects and may have great potential in clinical applications.

[0118] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing a bone regeneration scaffold capable of sequentially releasing drugs, comprising the following steps: Providing drug-loaded mesoporous silica microspheres; the drug is dexamethasone; mixing methacrylic anhydride gelatin, sodium alginate, a photoinitiator, hydroxyapatite, an inorganic copper salt and the drug-loaded mesoporous silica microspheres to obtain a bio-ink; A sacrificial bio-ink aqueous solution is used as the core layer material, and the bio-ink is used as the shell layer material. The sacrificial bio-ink aqueous solution and the bio-ink are coaxially extruded and printed and photocrosslinked to obtain a pre-crosslinked regenerative bone scaffold; the coaxial extrusion printing and photocrosslinking temperatures are independently 3-5°C; the sacrificial bio-ink material includes one or more of gelatin, polyoxyethylene polyoxypropylene ether, and polyethylene glycol; mixing the pre-crosslinked regenerative bone scaffold and a calcium salt solution, and performing re-crosslinking to obtain a primary regenerative bone scaffold; The sacrificial bio-ink material is removed from the primary regenerated bone scaffold, and the obtained bone scaffold with a hollow structure is sequentially frozen and freeze-dried to obtain the regenerated bone scaffold.

2. The preparation method according to claim 1, characterized in that The method for preparing the drug-loaded mesoporous silica microspheres comprises the following steps: The mesoporous silica microspheres and the drug solution are mixed, the obtained suspension is vacuum loaded, and then solid-liquid separation is performed to obtain the drug-loaded mesoporous silica microspheres.

3. The preparation method according to claim 1 or 2, characterized in that The mass ratio of the drug to the mesoporous silica microspheres is 1:300-400.

4. The preparation method according to claim 2, characterized in that The vacuum degree of the vacuum load is 8-10 kPa, the temperature is 20-30° C., and the time is 4-8 hours.

5. The preparation method according to claim 2, characterized in that The mesoporous silica microspheres have a diameter of 500-900 nm and a specific surface area of ​​300-600 m 2 / g.

6. The preparation method according to claim 1, characterized in that The photoinitiator includes one or more of lithium phenyl-2,4,6-trimethylbenzoylphosphite, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, 2,2'-azo(2-methyl-N-(2-hydroxyethyl)propionamide) and ethyl 2,4,6-trimethylbenzoylphenylphosphonate.

7. The preparation method according to claim 1 or 6, characterized in that The photocrosslinking is ultraviolet light crosslinking, the wavelength of the ultraviolet light is 400-410 nm, and the photocrosslinking time is 2-4 minutes.

8. The preparation method according to claim 1, characterized in that The concentration of the calcium salt solution is 8-12 wt %, the re-crosslinking temperature is 3-5° C., and the time is 25-35 min.

9. The regenerated bone scaffold prepared by the preparation method according to any one of claims 1 to 8, wherein the pore size between fibers of the regenerated bone scaffold is 400-600 μm and the porosity is 70-90%.

10. Use of the regenerative bone scaffold according to claim 9 in preparing a material for treating bone defects and repairing the bone.

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