Biological 3D intelligent response scaffold based on silk fibroin as well as preparation method and application of biological 3D intelligent response scaffold

Abstract

The invention relates to the technical field of biological scaffolds, in particular to a silk fibroin-based biological 3D intelligent response scaffold and a preparation method and application thereof.The silk fibroin-based biological 3D intelligent response scaffold adopts a three-layer bionic gradient structure of an oriented fiber membrane, a porous sponge layer and an interface layer, simulates natural tissues, and has the advantages that the bionic gradient structure is improved; mechanical guidance, cell inhabitation and biological activity interfaces are provided respectively, and high-quality regeneration of blood vessels, nerves and skin is promoted synergistically. According to the present invention, the pH / enzyme / temperature triple response unit is provided, such that the lesion microenvironment can be perceived, the targeted release can be triggered, the problem of high burst release rate of the traditional physical drug loading can be solved, and the on-demand accurate drug delivery can be achieved. The SF / HPMC gel is adopted for 3D printing, the printing precision is high, and the porosity difference is 1t; the product performance is stable, and the pore structure can be precisely designed and customized according to the regeneration requirements of different tissues (such as blood vessels, nerves and skin).

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological scaffolds, in particular to a silk fibroin-based biological 3D intelligent response scaffold and a preparation method and application thereof. BACKGROUND

[0002] Silk fibroin has been widely studied in the field of tissue engineering scaffolds due to its good biocompatibility, degradability and mechanical properties. In the prior art, various scaffolds based on silk fibroin have been developed. For example, Chinese patent CN107137774A discloses a silk fibroin / hydroxyapatite composite bone scaffold, but it introduces hydroxyapatite, which has the problems of high brittleness and easy breakage, limiting its application in flexible tissue repair. Another Chinese patent CN111823569A discloses an SF / HPMC (hydroxypropyl methyl cellulose) scaffold, which improves the processing performance through HPMC, but its structure is usually single, lacks biomimetic gradient design and intelligent drug release function, resulting in deficiencies in guiding directional regeneration of nerves, inhibiting thrombus formation and avoiding drug burst release, etc. Specifically, the shortcomings of the prior art mainly include: Single structure: Most scaffolds are single-layer homogeneous structures or simple double-layer structures, which cannot simulate the multi-layer heterogeneous structure of natural tissues (such as blood vessels and nerves), resulting in single function and inability to meet multiple requirements such as mechanical support, cell infiltration and barrier protection.

[0003] Poor drug release control: Drugs are usually loaded by physical mixing, and the release behavior is uncontrollable, with serious initial burst release (such as more than 50% within 24 hours), which cannot achieve targeted and on-demand drug delivery for pathological microenvironment (such as pH and specific enzyme concentration), affecting the therapeutic effect and possibly causing toxic side effects.

[0004] Poor consistency in preparation process: Traditional preparation methods (such as solvent casting / particle leaching) are difficult to accurately control the macroscopic morphology and microstructure (such as pore size and porosity) of the scaffold, with large differences between batches (porosity difference can exceed 20%), which is not conducive to large-scale production and clinical application.

[0005] Therefore, developing an integrated scaffold with biomimetic multi-layer structure, intelligent drug release control and high-precision manufacturing process is the key to solving the above technical bottlenecks. SUMMARY

[0006] The present application aims to overcome the shortcomings of the prior art and provide a silk fibroin-based biological 3D intelligent response scaffold and a preparation method and application thereof, which can simulate the complex structure of natural tissues, achieve targeted release of drugs in specific physiological environments, have excellent mechanical properties and biocompatibility, and can be prepared with high precision and in large scale.

[0007] The application aims to provide a silk fibroin-based biological 3D intelligent response scaffold. The directional fiber membrane is a directional fiber membrane prepared from silk fibroin (SF) and polycaprolactone (PCL) through electrospinning. The porous sponge layer is a porous sponge layer prepared from silk fibroin (SF) and hydroxypropyl methyl cellulose (HPMC) through 3D printing. The interface layer is a de-cellularized amniotic membrane (DHAM) interface layer.

[0008] Preferably, the porous sponge layer is loaded with an environment-responsive drug controlled release unit.

[0009] Preferably, the environment-responsive drug controlled release unit is a pH response unit, an enzyme response unit or a temperature response unit. The pH response unit comprises basic fibroblast growth factor (bFGF) loaded in carboxymethyl chitosan (CMC). The enzyme response unit comprises a heparin-vascular endothelial growth factor (VEGF) complex cross-linked by matrix metalloproteinase-2 (MMP-2) sensitive peptide. The temperature response unit comprises brain-derived neurotrophic factor (BDNF) wrapped in poloxamer F127.

[0010] Preferably, the fiber diameter of the directional fiber membrane ranges from 500 nm to 1000 nm, and the fiber orientation degree is greater than 90%.

[0011] Preferably, the porosity of the porous sponge layer is 60% to 75%, and the pore size is 50 to 1200 μm.

[0012] A preparation method of a silk fibroin-based biological 3D intelligent response scaffold comprises the following steps: Step (a): preparing a directional fiber membrane; silk fibroin (SF) and polycaprolactone (PCL) are prepared into a spinning solution, and then a directional fiber membrane is prepared through electrospinning; Step (b): preparing a porous sponge layer; SF / HPMC gel is prepared from silk fibroin (SF) and hydroxypropyl methyl cellulose (HPMC), a 3D printing scaffold blank is prepared, the directional fiber membrane is attached to the surface of the scaffold blank, and finally the porous sponge layer is prepared through freeze-drying; Step (c): attaching a de-cellularized amniotic membrane (DHAM) interface layer to the porous sponge layer, and then cross-linking through a cross-linking agent, namely genipin, to obtain a silk fibroin-based biological 3D intelligent response scaffold.

[0013] Preferably, step (b) further includes loading an environmentally responsive drug controlled-release unit in the porous sponge layer, specifically: subjecting the freeze-dried porous sponge layer to plasma treatment, then immersing it in a drug-containing solution at pH 7.0-pH 7.6, performing chemical cross-linking or physical adsorption to fix the drug at 35℃-39℃, and drying to obtain the porous sponge layer of the environmentally responsive drug controlled-release unit.

[0014] Preferably, during plasma treatment, the plasma is applied under oxygen conditions at a moderate power (e.g., 50-200W) with an intermittent glow discharge time of 5-10 minutes.

[0015] Preferably, in step (a), SF powder and PCL particles are mixed at a mass ratio of (7-9):(3-1), dissolved in hexafluoroisopropanol (HFIP), and magnetically stirred for 12-36 hours to prepare a spinning solution with a concentration of 11-13wt%.

[0016] Preferably, in step (a), the electrospun oriented fiber membrane is dried at 35-45°C and -0.10 to -0.08 MPa for 6-18 hours.

[0017] Preferably, in step (a), the electrospinning uses an inclined receiving roller with its axis at a 15° angle to the direction of the spinning nozzle, so that the fibers are oriented at a 70°-110° cross-axis along the long axis of the blood vessel.

[0018] Preferably, the molecular weight of the PCL particles used in this invention is 70,000-90,000.

[0019] Preferably, in step (b), SF powder is dissolved in deionized water and magnetically stirred at 55-65°C for 1-3 hours to prepare a 28-32 wt% SF solution. Then, hydroxypropyl methylcellulose HPMC is added at a mass ratio of SF:HPMC of (8-10):(2-1) and magnetically stirred at 2-8°C for 6-18 hours to obtain an SF / HPMC gel.

[0020] Preferably, the specific parameters for 3D printing in step (b) are as follows: nozzle diameter: 0.3mm, printing speed: 6mm / s, layer height: 100um, infill spacing: 1.4mm, infill path: diamond grid, printing platform temperature: 5℃.

[0021] Preferably, in step (b) freeze-drying, the support blank is first pre-frozen at -75 to -85°C for 3-5 hours, and then freeze-dried at a vacuum of ≤10Pa for 24-36 hours.

[0022] Preferably, the interface layer before bonding in step (c) needs to be pretreated: the decellularized amniotic membrane DHAM is immersed in 0.5%-1.0% (w / v) sodium dodecyl sulfate SDS aqueous solution and shaken at room temperature for 12-24 hours, then removed and washed, and then immersed in 1.0%-2.0% (v / v) polyethylene glycol octylphenyl ether Triton X-100 aqueous solution and shaken at room temperature for 12-24 hours, and then washed clean; Preferably, in step (c): the bonding pressure is 0.05-0.15 MPa during bonding, and it is maintained for 20-40 min. The bonded scaffold is then immersed in 0.1-0.2% genipin solution for crosslinking at 35-38℃ for 12-24 hours. After washing, it is dried at 35-38℃ to obtain a 3D intelligent responsive scaffold based on silk fibroin. The genipin solution is PBS buffer with pH = 7.4.

[0023] Preferably, the total thickness of the fibroin-based bio-3D intelligent responsive scaffold of the present invention is controlled within 1.8-2.0 mm.

[0024] The present invention features a three-layer biomimetic gradient structure: an electrospun SF / polycaprolactone (PCL) oriented fiber membrane (fiber orientation >95%) for nerve axon guidance or vascular anti-leakage; a 3D-printed SF / hydroxypropyl carboxymethyl cellulose (HPMC) porous sponge layer for loading stem cells or drugs; and a decellularized amnion (DHAM) interface layer to inhibit stone formation and promote epithelialization.

[0025] The multi-response drug controlled-release system of the present invention comprises: a pH-responsive unit: basic fibroblast growth factor (bFGF) encapsulated in carboxymethyl chitosan (CMC), which triggers release in the acidic environment of the wound (pH<6.5); an enzyme-responsive unit: a heparin-VEGF complex cross-linked with matrix metalloproteinase-2 (MMP-2) sensitive peptide, which dissociates when MMP-2 is overexpressed at the inflammatory site; and a temperature-responsive unit: neurotrophic factor (BDNF) encapsulated in poloxamer F127, which induces gel phase transition at body temperature (37°C) to achieve sustained release.

[0026] The integrated 3D printing process of this invention: The SF / HPMC gel combined with the low-temperature deposition modeling of 3D printing makes the porosity difference of this invention <2%, the product performance is stable, and the pore structure can be precisely designed and customized according to the regeneration needs of different tissues (such as blood vessels, nerves, and skin).

[0027] The present invention relates to the application of a silk fibroin-based 3D intelligent responsive scaffold in the preparation of tissue engineering products, wherein the tissue engineering products are vascular scaffolds, nerve repair catheters, or wound dressings.

[0028] The beneficial effects of this invention are as follows: The bio-3D intelligent responsive scaffold of this invention adopts a three-layer biomimetic gradient structure of directional fiber membrane, porous sponge layer, and interface layer, simulating natural tissue and providing mechanical guidance, cell habitat, and bioactive interface, respectively, synergistically promoting high-quality regeneration of blood vessels, nerves, and skin. This invention features a triple-response unit for pH / enzyme / temperature, capable of sensing the disease microenvironment and triggering targeted release, solving the problem of high burst release rate in traditional physical drug delivery systems, and achieving precise on-demand drug delivery. This invention uses SF / HPMC gel for 3D printing, achieving high printing precision, resulting in a porosity difference of <2%, stable product performance, and a pore structure that can be precisely designed and customized according to the regeneration needs of different tissues (such as blood vessels, nerves, and skin). Detailed Implementation

[0029] The present invention will be further described in conjunction with the following embodiments.

[0030] Example 1 A bio-3D smart responsive scaffold based on silk fibroin, comprising a directional fiber membrane, a porous sponge layer, and an interface layer, specifically: The oriented fiber membrane is an oriented fiber membrane made of silk fibroin (SF) and polycaprolactone (PCL) by electrospinning; The porous sponge layer is a porous sponge layer made of silk fibroin (SF) and hydroxypropyl methylcellulose (HPMC) through 3D printing; The interface layer is a decellularized amnion (DHAM) interface layer.

[0031] The fiber diameter in the oriented fiber membrane is 500 nm, and the fiber orientation degree is greater than 90%.

[0032] A method for preparing a silk fibroin-based bio-3D smart responsive scaffold includes the following steps: Step (a): Preparation of oriented fiber membrane; a spinning solution is prepared from silk fibroin (SF) and polycaprolactone (PCL), and then oriented fiber membrane is prepared by electrospinning; Step (b): Preparation of porous sponge layer: SF / HPMC gel is prepared by silk fibroin (SF) and hydroxypropyl methylcellulose (HPMC), scaffold blank is 3D printed, oriented fiber membrane is then attached to the surface of the scaffold blank, and finally freeze-drying is performed to prepare porous sponge layer; Step (c): The decellularized amnion (DHAM) interface layer is attached to the porous sponge layer and then cross-linked with the cross-linking agent genipin to obtain a biological 3D smart responsive scaffold based on silk fibroin.

[0033] In step (a), SF powder and PCL particles are mixed at a mass ratio of 7:3, dissolved in hexafluoroisopropanol (HFIP), and magnetically stirred for 12 hours to prepare a spinning solution with a concentration of 11wt%.

[0034] In step (a), the electrospun oriented fiber membrane needs to be dried at 35°C and -0.10 MPa for 6 hours.

[0035] In step (a), the electrospinning uses an inclined receiving roller with its axis at a 15° angle to the direction of the spinning nozzle, so that the fibers are oriented at a 70° cross along the long axis of the blood vessel.

[0036] The molecular weight of the PCL particles used in this invention is 70,000.

[0037] In step (b), SF powder is dissolved in deionized water and magnetically stirred at 55°C for 1 hour to prepare a 28wt% SF solution. Then, hydroxypropyl methylcellulose HPMC is added at a mass ratio of SF:HPMC of 8:2 and magnetically stirred at 2°C for 6 hours to obtain an SF / HPMC gel.

[0038] The specific parameters for 3D printing in step (b) are as follows: nozzle diameter: 0.3mm, printing speed: 6mm / s, layer height: 100um, infill spacing: 1.4mm, infill path: diamond grid, printing platform temperature: 5℃.

[0039] In step (b), the support blank is first pre-frozen at -75℃ for 3 hours, and then freeze-dried at a vacuum of ≤10Pa for 24 hours.

[0040] In step (c), the interface layer before bonding needs to be pretreated: the decellularized amniotic membrane DHAM is immersed in 0.5% (w / v) sodium dodecyl sulfate SDS solution and shaken at room temperature for 12 hours, then taken out and washed, and then placed in 1.0% (v / v) polyethylene glycol octylphenyl ether Triton X-100 solution and shaken at room temperature for 12 hours, and then washed clean. In step (c): the bonding pressure is 0.05 MPa and maintained for 20 min. The bonded scaffold is then immersed in 0.1% genipin solution and crosslinked at 35°C for 24 hours. After cleaning, it is dried at 35°C to obtain a biological 3D intelligent responsive scaffold based on silk fibroin.

[0041] The total thickness of the fibroin-based bio-3D smart responsive scaffold of this invention is controlled at 1.8 mm.

[0042] Example 2

[0043] A bio-3D smart responsive scaffold based on silk fibroin, comprising a directional fiber membrane, a porous sponge layer, and an interface layer, specifically: The oriented fiber membrane is an oriented fiber membrane made of silk fibroin (SF) and polycaprolactone (PCL) by electrospinning; The porous sponge layer is a porous sponge layer made of silk fibroin (SF) and hydroxypropyl methylcellulose (HPMC) through 3D printing; The interface layer is a decellularized amnion (DHAM) interface layer.

[0044] The fiber diameter in the oriented fiber membrane is in the range of 800 nm, and the fiber orientation degree is greater than 90%.

[0045] A method for preparing a silk fibroin-based bio-3D smart responsive scaffold includes the following steps: Step (a): Preparation of oriented fiber membrane; a spinning solution is prepared from silk fibroin (SF) and polycaprolactone (PCL), and then oriented fiber membrane is prepared by electrospinning; Step (b): Preparation of porous sponge layer: SF / HPMC gel is prepared by silk fibroin (SF) and hydroxypropyl methylcellulose (HPMC), scaffold blank is 3D printed, oriented fiber membrane is then attached to the surface of the scaffold blank, and finally freeze-drying is performed to prepare porous sponge layer; Step (c): The decellularized amnion (DHAM) interface layer is attached to the porous sponge layer and then cross-linked with the cross-linking agent genipin to obtain a biological 3D smart responsive scaffold based on silk fibroin.

[0046] In step (a), SF powder and PCL particles are mixed at a mass ratio of 8:2, dissolved in hexafluoroisopropanol (HFIP), and magnetically stirred for 24 hours to prepare a spinning solution with a concentration of 12wt%.

[0047] In step (a), the electrospun oriented fiber membrane needs to be dried at 40°C and -0.09 MPa for 10 hours.

[0048] In step (a), the electrospinning uses an inclined receiving roller with its axis at a 15° angle to the direction of the spinning nozzle, so that the fibers are oriented at a 90° cross along the long axis of the blood vessel.

[0049] The molecular weight of the PCL particles used in this invention is 80,000.

[0050] In step (b), SF powder is dissolved in deionized water and magnetically stirred at 60°C for 2 hours to prepare a 30wt% SF solution. Then, hydroxypropyl methylcellulose HPMC is added at a mass ratio of SF:HPMC of 9:1 and magnetically stirred at 4°C for 12 hours to obtain an SF / HPMC gel.

[0051] The specific parameters for 3D printing in step (b) are as follows: nozzle diameter: 0.3mm, printing speed: 6mm / s, layer height: 100um, infill spacing: 1.4mm, infill path: diamond grid, printing platform temperature: 5℃.

[0052] In step (b), the support blank is first pre-frozen at -80℃ for 4 hours, and then freeze-dried at a vacuum of ≤10Pa for 30 hours.

[0053] In step (c), the interface layer before bonding requires pretreatment: the decellularized amniotic membrane DHAM is immersed in 0.8% (w / v) sodium dodecyl sulfate SDS solution and shaken at room temperature for 18 hours, then removed and washed, and then placed in 1.5% (v / v) polyethylene glycol octylphenyl ether Triton X-100 solution and shaken at room temperature for 18 hours, and then washed clean. In step (c): the bonding pressure is 0.1 MPa during bonding and is maintained for 30 min. The bonded scaffold is then immersed in 0.2% genipin solution and crosslinked at 36°C for 18 hours. After cleaning, it is dried at 36°C to obtain a biological 3D intelligent responsive scaffold based on silk fibroin.

[0054] The total thickness of the fibroin-based bio-3D intelligent responsive scaffold of this invention is controlled at 1.9 mm.

[0055] Example 3 A bio-3D smart responsive scaffold based on silk fibroin, comprising a directional fiber membrane, a porous sponge layer, and an interface layer, specifically: The oriented fiber membrane is an oriented fiber membrane made of silk fibroin (SF) and polycaprolactone (PCL) by electrospinning; The porous sponge layer is a porous sponge layer made of silk fibroin (SF) and hydroxypropyl methylcellulose (HPMC) through 3D printing; The interface layer is a decellularized amnion (DHAM) interface layer.

[0056] The fiber diameter in the oriented fiber membrane is in the range of 1000 nm, and the fiber orientation degree is greater than 90%.

[0057] A method for preparing a silk fibroin-based bio-3D smart responsive scaffold includes the following steps: Step (a): Preparation of oriented fiber membrane; a spinning solution is prepared from silk fibroin (SF) and polycaprolactone (PCL), and then oriented fiber membrane is prepared by electrospinning; Step (b): Preparation of porous sponge layer: SF / HPMC gel is prepared by silk fibroin (SF) and hydroxypropyl methylcellulose (HPMC), scaffold blank is 3D printed, oriented fiber membrane is then attached to the surface of the scaffold blank, and finally freeze-drying is performed to prepare porous sponge layer; Step (c): The decellularized amnion (DHAM) interface layer is attached to the porous sponge layer and then cross-linked with the cross-linking agent genipin to obtain a biological 3D smart responsive scaffold based on silk fibroin.

[0058] In step (a), SF powder and PCL particles are mixed at a mass ratio of 9:1, dissolved in hexafluoroisopropanol (HFIP), and magnetically stirred for 36 hours to prepare a spinning solution with a concentration of 13wt%.

[0059] In step (a), the electrospun oriented fiber membrane needs to be dried at 45°C and -0.08MPa for 18 hours.

[0060] In step (a), the electrospinning uses an inclined receiving roller with its axis at a 15° angle to the direction of the spinning nozzle, so that the fibers are oriented at a 110° cross along the long axis of the blood vessel.

[0061] The molecular weight of the PCL particles used in this invention is 90,000.

[0062] In step (b), SF powder is dissolved in deionized water and magnetically stirred at 65°C for 1 hour to prepare a 32wt% SF solution. Then, hydroxypropyl methylcellulose HPMC is added at a mass ratio of SF:HPMC of 10:1 and magnetically stirred at 8°C for 6 hours to obtain an SF / HPMC gel.

[0063] The specific parameters for 3D printing in step (b) are as follows: nozzle diameter: 0.3mm, printing speed: 6mm / s, layer height: 100um, infill spacing: 1.4mm, infill path: diamond grid, printing platform temperature: 5℃.

[0064] In step (b), the support blank is first pre-frozen at -85℃ for 3 hours, and then freeze-dried at a vacuum of ≤10Pa for 36 hours.

[0065] Among them, step (c) requires pretreatment of the interface layer before bonding: the decellularized amniotic membrane DHAM is immersed in 1.0% (w / v) sodium dodecyl sulfate SDS solution and shaken at room temperature for 24 hours, then taken out and washed, and then placed in 2.0% (v / v) polyethylene glycol octylphenyl ether Triton X-100 solution and shaken at room temperature for 24 hours, and then washed clean. In step (c): the bonding pressure is 0.15 MPa and maintained for 40 min. The bonded scaffold is then immersed in 0.2% genipin solution and crosslinked at 38°C for 12 hours. After cleaning, it is dried at 38°C to obtain a biological 3D intelligent responsive scaffold based on silk fibroin.

[0066] The total thickness of the fibroin-based bio-3D smart responsive scaffold of this invention is controlled at 2.0 mm.

[0067] Experimental data 1. Temperature properties of SF / HPMC gel The viscosity change of the SF / HPMC gel prepared in step (b) of Examples 1-3 of the present invention was measured using a rotational rheometer at 25°C. The results are shown in Table 1. Table 1 shows that the viscosity of the SF / HPMC gel of the present invention is controlled at 6500-7500 cP to ensure extrudability during 3D printing. After storage at 4°C for 72 hours, the viscosity change rate was measured to be < 5%, ensuring the stability of the printing process.

[0068] Table 1

[0069] 2. Determination of pore size and porosity of porous sponge layer The porous sponge layer prepared by freeze-drying in step (b) of Examples 1-3 of the present invention was measured by laser confocal microscopy. Fifty pores were randomly selected and statistically detected. The pore size was 207±1.66μm. The porosity was determined by the water displacement method. Table 2 shows the porosity experimental data record. It can be seen that the porosity of Examples 1-3 of the present invention is 68±2%.

[0070] Table 2

[0071] 3. MTT cytotoxicity assay Cytotoxicity was tested using the MTT assay. Table 3 shows the raw data for cytotoxicity of SF / HPMC gel and blank carrier scaffold, and Table 4 shows the cell viability data of SF / HPMC gel compared with blank carrier scaffold.

[0072] As shown in Table 4, the cell viability of all concentrations of SF / HPMC gel and blank carrier scaffold in the experimental group was significantly higher than 100% (minimum 114.30%, maximum 181.61%). A cell viability > 70% is considered to indicate no cytotoxicity. In the negative group, the viability ranged from 115% to 131%, slightly higher than the theoretical 100%, which is within normal fluctuations and confirms that the cells grew well in this experimental system, making the baseline reliable. In the positive group, the viability was effectively suppressed below 60%, demonstrating that the experimental system can accurately detect toxicity. The MTT cytotoxicity assay of this invention shows that the cell viability of the SF / HPMC gel and scaffold extract prepared in this invention was significantly higher than 100% at all tested concentrations, and far exceeded the 70% non-toxicity threshold, proving that the material has good biocompatibility.

[0073] Table 3

[0074] Table 4

[0075] Application Example 1: Application of Vascular Stents Based on Example 2, this application example loads the porous sponge layer of step (b) with an environmentally responsive drug controlled-release unit—an enzyme-responsive unit. Specifically: (1) A heparin-VEGF complex with a mass ratio of 1:0.5 and an MMP-2 sensitive peptide are mixed at a mass ratio of 10:1 and dissolved in PBS buffer (pH=7.4) to obtain a drug-containing solution; (2) The freeze-dried porous sponge layer is subjected to plasma treatment, and then immersed in a drug-containing solution at pH 7.0-pH 7.6. The drug is fixed by chemical cross-linking at 37°C for 2 hours using EDC / NHS catalysis, so that the amino group of the sensitive peptide forms an amide bond with the carboxyl group of SF on the sponge surface; the layer is washed and vacuum dried (37°C, 4 hours) to obtain a porous sponge layer loaded with an environmentally responsive drug controlled-release unit, with a drug loading of 15ug / mg. Other parts are the same as in Example 2.

[0076] Finally, in Application Example 1, a 3D intelligent responsive scaffold based on silk fibroin was implanted into the abdominal aorta of a rat (4 mm in diameter). The results showed that the patency rate was >90% after 6 months (compared to 60% for traditional ePTFE scaffolds); the endothelialization coverage reached 95% after 7 days (RGD peptide-modified surface). For a detailed comparison of performance indicators, please refer to Table 5.

[0077] Table 5

[0078] Application Example 2: Application of Nerve Repair Catheters Based on Example 2, this application example loads the porous sponge layer of step (b) with an environmentally responsive drug controlled-release unit—a temperature-responsive unit. Specifically: (1) Poloxamer F127 powder is dissolved in phosphate-buffered saline (PBS) at low temperature (4°C) to obtain a 25% w / v low-temperature solution, which is a free-flowing liquid; (2) BDNF is added to the above low-temperature solution at 20 µg / mL and stirred at 4°C to disperse it evenly, forming a drug-loaded temperature-sensitive solution; (3) The freeze-dried porous sponge layer is subjected to plasma treatment, then immersed in the drug-loaded temperature-sensitive solution, placed in a vacuum desiccator, and briefly evacuated at a vacuum degree of -0.08 MPa for 10 minutes. The low-temperature liquid drug-loaded temperature-sensitive solution is drawn in by the pressure difference and fills the micropores of the porous sponge layer. It is then transferred to 37°C for gelation to obtain a porous sponge layer loaded with an environmentally responsive drug controlled-release unit. Other parts are the same as in Example 2.

[0079] Finally, in Application Example 2, a silk fibroin-based 3D intelligent responsive scaffold (catheter) was implanted into a 10 mm sciatic nerve defect in rats. After 12 weeks, the nerve conduction velocity recovery rate was 85%, comparable to autologous transplantation. Poloxamer F127, loaded with BDNF, was continuously released at 37°C for 14 days, promoting Schwann cell migration (S-100+ expression increased 3-fold). Detailed data on BDNF continuous release over 14 days are shown in Table 6, and the quantification data of cell function (S-100+ expression and migration ability) are shown in Table 7. Table 7 shows no significant difference between the vector-only group and the control group (P>0.05), proving that BDNF is the core factor in activating S-100 expression in Schwann cells, and the 3-fold increase directly reflects the cell function activation effect. The migration speed and distance of the experimental group were nearly twice that of the control group, and the difference was extremely significant (P<0.01), indicating that continuously released BDNF can effectively drive Schwann cells to migrate to the injury site, providing a cellular basis for nerve regeneration.

[0080] Table 6

[0081] Table 7

[0082] Application Example 3: Application of Wound Dressings Based on Example 2, this application example loads an environmentally responsive drug-controlled release unit—a pH-responsive unit—on the porous sponge layer in step (b). Specifically: (1) Carboxymethyl chitosan (CMC) is dissolved in water to obtain a 2% w / v transparent viscous solution; (2) Basic fibroblast growth factor (bFGF) is added to the CMC solution at 50 μg / mL, stirred and mixed to obtain a drug-loaded solution; (3) The freeze-dried porous sponge layer is subjected to plasma treatment, then immersed in the drug-loaded solution, placed in a vacuum dryer and briefly evacuated at a vacuum degree of -0.08 MPa for 10 minutes. The low-temperature liquid drug-loaded solution is drawn in by the pressure difference and fills the micropores of the porous sponge layer. The layer is then removed and dried to obtain a porous sponge layer loaded with an environmentally responsive drug-controlled release unit. Other parts are the same as in Example 2.

[0083] Finally, in Application Example 3, a 3D intelligent responsive scaffold based on silk fibroin was used on an infected wound. At pH 5.5 (infected wound), the release rate was >80% after 48 hours, accelerating epithelial regeneration. The release rate of bFGF on the wound is shown in Table 8. Table 8 shows an overall pattern of "rapid release in the early stage (release rate 2.95%-4.25% / h in the 0-12h range), gradual release in the middle stage (release rate 2.05%-2.95% / h in the 12-24h range), and slow release in the later stage (release rate 0.15%-1.45% / h in the 24-48h range)," consistent with the common kinetic characteristics of enzyme-responsive or pH-responsive drug release. The rapid release in the early stage quickly activates epithelial regeneration, while the slow release in the later stage maintains local drug concentration and continuously promotes healing. In the early stage (0-24h) when the release rate is high, epithelial regeneration is mainly characterized by cell activation, migration and initial aggregation; in the middle and late stages (24-48h) when the release rate slows down, epithelial regeneration enters the stage of cell differentiation and lamellar structure formation. The release process is highly matched with the regeneration stage, which reflects the precision of pH-responsive release.

[0084] Table 8

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A silk fibroin-based bio-3D smart response scaffold, characterized in that, The biological 3D intelligent response stent comprises a directional fiber membrane, a porous sponge layer and an interface layer, in particular: The directional fiber membrane is a directional fiber membrane made of silk fibroin and polycaprolactone by electrospinning. The porous sponge layer is a porous sponge layer made of silk fibroin and hydroxypropyl methylcellulose by 3D printing. The interface layer is a decellularized amniotic membrane interface layer.

2. The silk fibroin-based bio-3D smart response scaffold according to claim 1, characterized in that, The porous sponge layer is loaded with an environment-responsive drug controlled release unit.

3. The silk fibroin-based bio-3D smart response scaffold according to claim 2, characterized in that, The environment-responsive drug controlled release unit is a pH-responsive unit, an enzyme-responsive unit or a temperature-responsive unit, in particular: The pH-responsive unit comprises basic fibroblast growth factor loaded in carboxymethyl chitosan; The enzyme-responsive unit comprises a heparin-vascular endothelial growth factor complex cross-linked by matrix metalloproteinase-2 sensitive peptide; The temperature-responsive unit comprises brain-derived neurotrophic factor wrapped in poloxamer.

4. The silk fibroin-based bio-3D smart response scaffold according to claim 1, characterized in that, The fiber diameter of the directional fiber membrane ranges from 500 nm to 1000 nm, and the fiber orientation degree is greater than 90%.

5. The silk fibroin based bio 3D smart response scaffold according to claim 1, wherein, The porosity of the porous sponge layer is 60% to 75%, and the pore size is 50 to 1200 μm.

6. A method for preparing a silk fibroin-based biological 3D smart response scaffold, characterized in that, The method comprises the following steps: Step (a): preparing a directional fiber membrane; Prepare a spinning solution from silk fibroin and polycaprolactone, and then prepare a directional fiber membrane by electrospinning; Step (b): preparing a porous sponge layer: prepare an SF / HPMC gel from silk fibroin and hydroxypropyl methylcellulose, print a scaffold blank by 3D printing, then adhere the directional fiber membrane to the surface of the scaffold blank, and finally freeze-dry to prepare a porous sponge layer; Step (c): adhere the decellularized amniotic membrane interface layer to the porous sponge layer, and then cross-link it with the cross-linking agent genipin to obtain a silk fibroin-based biological 3D intelligent response stent.

7. The method according to claim 6, wherein the method is characterized by, The step (b) further comprises loading the environment-responsive drug controlled release unit in the porous sponge layer, specifically: after freeze-drying the porous sponge layer, perform plasma treatment, then soak it in a drug-containing solution, perform chemical cross-linking or physical adsorption to fix the drug, and dry to obtain the environment-responsive drug controlled release unit porous sponge layer.

8. The method according to claim 6, wherein the method is characterized by, In step (a), the SF powder and PCL particles are mixed in a mass ratio of (7-9):(3-1), dissolved in hexafluoroisopropanol, and magnetically stirred for 12-36 h to prepare a spinning solution with a concentration of 11-13 wt%.

9. The method of claim 6, wherein the method is characterized by the steps of: In step (b), the SF powder is dissolved in deionized water, magnetically stirred at 55-65°C for 1-3 h to prepare a 28-32 wt% SF solution, then HPMC is added in a mass ratio of (8-10):(2-1), and magnetically stirred at 2-8°C for 6-18 h to obtain an SF / HPMC gel.

10. Use of a silk fibroin-based bio-3D smart response scaffold according to any one of claims 1-9 for the preparation of a tissue engineering product, characterized in that, The tissue engineering product is a vascular stent, a nerve repair conduit or a wound dressing.

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