CN116392647B - Silk fibroin-based three-dimensional structure bilayer membrane for periodontal regeneration and preparation method and application thereof - Google Patents

Abstract

The present invention provides a silk-based three-dimensional structure double-layer membrane for periodontal regeneration, and a preparation method and application thereof, wherein the silk-based three-dimensional structure double-layer membrane for periodontal regeneration comprises an inner porous loose layer and an outer dense layer, wherein the porous loose layer is a nanofiber membrane composed of silk fibroin and nano-hydroxyapatite, and the dense layer is a nanofiber membrane composed of silk fibroin and biodegradable materials, and a cross-linked structure is formed between the porous loose layer and the dense layer. The silk-based three-dimensional structure double-layer membrane of the present invention has a three-dimensional structure, has a good mechanical barrier effect, can induce bone tissue regeneration, has a soft membrane structure, is well attached to periodontal tissue, has good mechanical properties and degradation properties, has the advantages of simple preparation method operation and mild experimental preparation conditions, and is suitable for large-scale batch production.

Description

A silk-based three-dimensional double-layer membrane for periodontal regeneration and its preparation method and application

Technical Field

The invention belongs to the field of preparation of nanofiber double-layer membranes, and relates to a silk-based three-dimensional structure double-layer membrane for periodontal regeneration, and a preparation method and application thereof.

Background technique

Oral implant refers to the use of biomaterials to make artificial roots, crowns, etc. to repair missing teeth and surrounding tissues, and achieve long-term stable and comfortable chewing function and tooth appearance. However, after tooth loss, the alveolar bone often gradually shrinks due to factors such as loss of functional stimulation, resulting in the common problem of insufficient bone mass in implant restoration, which has become one of the main challenges faced by this field. Therefore, before performing oral implant surgery, the patient's bone quality needs to be evaluated. For patients with less alveolar bone mass, bone grafting surgery is required first, using technological regenerative materials such as bone powder and periosteum to replace human bone. This technology is called guided bone regeneration (GBR). During the entire implant restoration process, some patients need bone augmentation surgery, and some of them need bone powder and oral repair membranes. The use of oral repair membrane materials in dental implants can promote bone regeneration, improve implant stability, and ensure good development of bone and grafted bone. It has good therapeutic effects and is of great value in improving patients' oral health and quality of life.

By covering the bone defect with an oral repair membrane, a mechanical barrier can be formed to achieve a sealing effect. This covering can relieve the pressure of the covering tissue, protect the blood clot, and form a good bone formation space. The barrier can block fibroblasts and connective tissue that affect bone formation, allowing precursor osteoblasts with growth potential to enter the bone defect area, thereby inducing bone repair and regeneration in the bone defect area and increasing the success rate of oral implant restoration.

There are many types of oral repair membranes. Traditional oral repair membranes made of alloy materials and polymer materials are gradually being eliminated due to their poor biocompatibility and non-degradability. Collagen oral repair membranes currently occupy the main market, but similar products on the market have more or less some defects:

(1) Collagen degrades quickly and cannot maintain its spatial structure in the later stages of repair;

(2) The dense surface cannot prevent soft tissue from invading the bone defect area, and the expected effect of the barrier membrane cannot be achieved;

(3) The porous surface has poor permeability and cannot effectively lock in the blood seepage, resulting in poor induction of bone regeneration;

(4) The oral repair membrane feels stiff and has poor adhesion, which will affect the convenience of surgical operation.

In contrast, silk fibroin (SF), as a natural polymer material, has good biodegradability, mechanical strength and biocompatibility, and is widely used in tissue engineering of bone, cartilage, liver and skin. However, the poor adhesion and hydrophilicity of silk fibroin limit its application in medical materials. After cross-linking modification, silk fibroin can induce the formation of β-folding structure, thereby improving the cell adhesion and elastic modulus of the material. Electrospinning technology can be used to prepare nano-scale silk fibroin fiber mesh, which has high mechanical strength and is closer to the physical structure of natural periosteum. Electrospun monolayer membranes have the disadvantages of thin thickness, poor strength, and easy swelling in water.

Therefore, in the art, it is desired to develop a material that can have ideal tissue conformability, mechanical properties and degradation cycle.

Summary of the invention

In view of the deficiencies of the prior art, the object of the present invention is to provide a silk-based three-dimensional structured double-layer membrane for periodontal regeneration, and a preparation method and application thereof.

In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

On the one hand, the present invention provides a silk-based three-dimensional structure double-layer membrane for periodontal regeneration, which includes an inner porous loose layer and an outer dense layer, wherein the porous loose layer is a nanofiber membrane composed of silk fibroin and nano-hydroxyapatite, and the dense layer is a nanofiber membrane composed of silk fibroin and biodegradable materials, and a cross-linked structure is formed between the porous loose layer and the dense layer.

In the present invention, silk fibroin is compounded with nano-hydroxyapatite (nHA) to increase the surface roughness and greatly improve the mechanical strength. In addition, the addition of polymer biodegradable materials can further increase the stability and mechanical strength of the nanofiber membrane. The porous loose layer and the outer dense layer form a cross-linked structure, which can induce silk fibroin to form a β-folded structure, so that the silk-based double-layer membrane for periodontal regeneration of the present invention has a three-dimensional structure, which has more ideal tissue adhesion, mechanical properties, and degradation cycle than collagen membranes, and has great prospects in clinical applications.

Preferably, the mass proportion of nano-hydroxyapatite in the porous loose layer is 10% to 30%, for example, 10%, 13%, 15%, 18%, 20%, 23%, 25%, 28% or 30%.

Preferably, the particle size of the nano-hydroxyapatite is 20-40 nm, for example, 20 nm, 23 nm, 25 nm, 28 nm, 30 nm, 35 nm, 38 nm or 40 nm. Since the silk-based three-dimensional structured double-layer membrane for periodontal regeneration of the present invention is prepared by electrospinning, the particle size of the nano-hydroxyapatite is maintained at 20-40 nm by grinding and sieving, so that it is better dispersed in the electrospinning solution to avoid sedimentation; at the same time, it is not easy to block the needle during the electrospinning process, making the spinning process smoother.

Preferably, the weight percentage of silk fibroin in the dense layer is 50% to 100%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

Preferably, the biodegradable material is selected from any one of polylactic acid, polycaprolactone or polyhydroxybutyrate-valeric acid copolyester or a combination of at least two thereof.

Preferably, the crosslinking agent used in the crosslinking is dicarbodiimide salt (EDC) and hydroxysuccinimide (NHS).

Preferably, the mass ratio of the carbodiimide salt to hydroxysuccinimide is 20% to 30%, for example, 20%, 23%, 25%, 28% or 30%.

In the present invention, during the cross-linking process of the porous loose layer and the dense layer, silk fibroin can be induced to form a β-folded structure, and the formed silk-based ultra-thin double-layer membrane has better adhesion to the periodontal tissue. In comparison, the commercially available double-layer collagen membrane feels stiff and has poor adhesion effect.

On the other hand, the present invention provides a method for preparing the silk-based three-dimensional structured double-layer membrane for periodontal regeneration as described above, the preparation method comprising the following steps:

(1) using a solution containing silk fibroin and nano-hydroxyapatite as an electrospinning solution, and obtaining a porous loose layer by electrospinning;

(2) using a solution containing silk fibroin and biodegradable materials as an electrospinning solution, and receiving and obtaining a dense layer on the obtained porous loose layer through electrospinning;

(3) Using a cross-linking agent to cross-link the porous loose layer and the dense layer to obtain the silk-based three-dimensional structure double-layer membrane for periodontal regeneration.

In the present invention, a dense layer of silk fibroin and biodegradable material is formed on the porous loose layer by electrospinning, and then the silk fibroin is induced to form a β-folded structure by cross-linking. The formed silk-based ultra-thin double-layer membrane has better adhesion to the periodontal tissue. In comparison, the commercially available double-layer collagen membrane feels stiff and has poor adhesion effect.

The present invention combines natural polymer material silk fibroin (SF), biodegradable material and inorganic component nanohydroxyapatite (nHA) to form a composite material, with complementary advantages, to construct a silk-based three-dimensional double-layer membrane. The silk fibroin membrane, as the main material, has good biodegradability and biocompatibility; the biodegradable material, with good biocompatibility and good biodegradability, can be used together with nanohydroxyapatite as an auxiliary material to improve the shortcomings of insufficient mechanical properties of silk fibroin nanofibers, while improving surface roughness and bone induction performance.

The present invention carries out the synthesis design of biomaterials based on the research ideas of material optimization design and biomimetic periosteum structure, and prepares a porous loose membrane by spinning the natural material silk fibroin and nano-hydroxyapatite composite, and then spins the silk fibroin/poly-L-lactide-caprolactone dense membrane on it, and then cross-links it to construct a silk fibroin-based three-dimensional double-layer membrane with biomimetic periosteum structure. The outer layer of the regeneration membrane is dense, which blocks the growth of fibroblasts; the inner layer is loose, which is conducive to the adhesion and proliferation of osteoblasts.

Preferably, the total mass percentage of silk fibroin and nano-hydroxyapatite in the solution containing silk fibroin and nano-hydroxyapatite in step (1) is 15% to 25%, for example, 15%, 18%, 20%, 22%, 24% or 25%.

Preferably, the solvent of the solution containing silk fibroin and nano-hydroxyapatite in step (1) is hexafluoroisopropanol (HFIP).

Preferably, the preparation process of the solution containing silk fibroin and nano-hydroxyapatite in step (1) is: adding freeze-dried silk fibroin and nano-hydroxyapatite particles to hexafluoroisopropanol to obtain a mixed solution, treating the mixed solution with ultrasonic vibration, and then stirring to obtain the solution containing silk fibroin and nano-hydroxyapatite.

Preferably, the ultrasonic power during the ultrasonic oscillation treatment is 19-23 KHz, for example, 19 KHz, 20 KHz, 21 KHz, 22 KHz or 23 KHz, and the treatment time is 10-40 min, for example, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min or 40 min.

In the present invention, the stirring as mentioned above may be magnetic stirring, and the stirring time is 3-48 hours.

Preferably, during the electrospinning in step (1), the voltage is set to 10-20 kV (for example, 10 kV, 13 kV, 15 kV, 18 kV or 20 kV), the feed rate is set to 0.8-1.5 mL/h (for example, 0.8 mL/h, 1.0 mL/h, 1.2 mL/h or 1.5 mL/h), the fiber is received by a roller, the roller speed is 800-3000 r/min (for example, 800 r/min, 1000 r/min, 1300 r/min, 1500 r/min, 2000 r/min, 2500 r/min or 3000 r/min), and the receiving distance is 10-15 cm (for example, 10 cm, 11 cm, 12 cm, 13 cm, 14 cm or 15 cm).

In the present invention, the electrospinning solution in step (1) is transferred into a 10 mL medical syringe in batches, a 20G needle is connected to the syringe, and then the syringe is placed in the propulsion pump of the electrospinning device.

Preferably, the electrospinning in step (1) is carried out at room temperature, and the air humidity is 15-20%, such as 15%, 17%, 18%, 19% or 20%.

Preferably, the mass percentage of silk fibroin and biodegradable material in the solution containing silk fibroin and biodegradable material in step (2) is 10% to 20%, for example, 10%, 13%, 15%, 18% or 20%.

Preferably, the solvent of the solution containing silk fibroin and biodegradable material in step (2) is at least one of hexafluoroisopropanol (HFIP), trifluoroacetic acid (TFA), acetic acid (HAc) or tetrahydrofuran (THF).

Preferably, the preparation process of the solution containing silk fibroin and biodegradable material in step (2) is: adding silk fibroin and biodegradable material to hexafluoroisopropanol to obtain a mixed solution, stirring to obtain the solution containing silk fibroin and biodegradable material.

In the present invention, the stirring as described above can be magnetic stirring, and the stirring time is 3-24 hours. Preferably, during the electrostatic spinning in step (2), the voltage is set to 10-20 kV (e.g., 10 kV, 13 kV, 15 kV, 18 kV or 20 kV), the injection rate is set to 0.8-1.5 mL/h (e.g., 0.8 mL/h, 1.0 mL/h, 1.2 mL/h or 1.5 mL/h), and the fiber is received by a drum, and the drum speed is 800-3000 r/min (e.g., 800 r/min, 1000 r/min, 1300 r/min, 1500 r/min, 2000 r/min, 2500 r/min or 3000 r/min), and the receiving distance is 10-15 cm (e.g., 10 cm, 11 cm, 12 cm, 13 cm, 14 cm or 15 cm).

In the present invention, the electrospinning solution in step (2) is transferred in batches into a 10 mL medical syringe, a 20G needle is connected to the syringe, and then the syringe is placed in the propulsion pump of the electrospinning device.

Preferably, the electrospinning in step (2) is carried out at room temperature, and the air humidity is 15-20%, such as 15%, 17%, 18%, 19% or 20%.

Preferably, the cross-linking agent in step (3) is diimide carbohydrate (EDC) and hydroxysuccinimide (NHS).

Preferably, the mass ratio of the carbodiimide salt to hydroxysuccinimide is 20% to 30%, for example, 20%, 23%, 25%, 28% or 30%.

Preferably, the cross-linking in step (3) is carried out at room temperature, and the cross-linking time is 1-6 hours, for example 1 hour, 2 hours, 3 hours, 4 hours, 5 hours or 6 hours.

Preferably, the crosslinking in step (3) is carried out in an organic solvent, and the organic solvent is anhydrous ethanol.

Preferably, in step (3), before cross-linking, the double-layer membrane obtained in step (2) is dried to remove the organic solvent remaining on the membrane.

After cross-linking in step (3), the residual cross-linking agent is rinsed with deionized water and then placed in a freeze dryer for drying.

On the other hand, the present invention provides the use of the silk-based three-dimensional structured double-layer membrane for periodontal regeneration as described above in the preparation of oral implant materials.

Compared with the prior art, the present invention has the following beneficial effects:

In the present invention, silk fibroin is compounded with nano-hydroxyapatite (nHA) to increase surface roughness and greatly improve mechanical strength. In addition, adding high molecular biodegradable materials can further increase the stability and mechanical strength of nanofiber membranes. The porous loose layer and the outer dense layer form a cross-linked structure, which can induce silk fibroin to form a β-folded structure, so that the silk fibroin-based double-layer membrane of periodontal regeneration of the present invention has a three-dimensional structure. As an oral repair membrane, its dense layer has a good mechanical barrier effect, and the loose layer plays an inducing bone tissue regeneration effect. While realizing the double-layer structure, it still has the physical properties of "ultra-thin", and the membrane structure is softer and better fits the periodontal tissue. It is more convenient to operate during clinical surgery, and it still has good mechanical properties and degradation performance while realizing the ultra-thin structure. And the preparation method of the present invention is simple to operate, and the advantages of mild experimental preparation conditions are suitable for large-scale batch production, and have good application prospects.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram and scanning electron microscope image of the structure of the silk-based three-dimensional structure double-layer membrane prepared in Example 3, wherein (A) a schematic diagram of the structure of the silk-based three-dimensional structure double-layer membrane, which includes a loose surface (wherein the small spheres represent nanohydroxyapatite) and a dense surface, (B) a scanning electron microscope image of the inner layer nHA@SF nanofiber membrane, with a scale of 10 μm, (C) a scanning electron microscope image of the outer layer 5SF/5P nanofiber membrane, with a scale of 10 μm, (D) a scanning electron microscope image of the cross section of the nHA@SF-5SF/5P double-layer nanofiber membrane, with a scale of 10 μm;

FIG2 is the infrared spectra of the films prepared in Example 3, (A) the infrared spectrum of the porous loose film nHA@SF, (B) the infrared spectrum of the dense film 5SF/5P;

FIG3 is an X-ray diffraction pattern of the film prepared in Example 3;

FIG4 is a contact angle test result of the film prepared in Example 3;

FIG5 is a graph showing the mechanical properties test results of the silk-based three-dimensional structured double-layer membrane prepared in Examples 1-3, (A) stress-strain curve, (B) breaking strength test result graph, (C) breaking elongation test result graph, and (D) Young's modulus test result graph;

Figure 6 is a scanning electron microscope image of the inner nanofiber membrane prepared in Example 3, Example 4, and Example 5, wherein (A) is a scanning electron microscope image of the inner nanofiber membrane prepared in Example 3, with a scale of 10 μm, (B) is a scanning electron microscope image of the inner nanofiber membrane prepared in Example 4, with a scale of 10 μm, and (C) is a scanning electron microscope image of the inner nanofiber membrane prepared in Example 5, with a scale of 10 μm;

FIG7 is an in vitro degradation curve of the membranes prepared in Example 2 and Comparative Example 1;

FIG8 is a scanning electron microscope image of the films prepared in Example 2 and Comparative Example 2;

FIG. 9 is a surface photograph of the films prepared in Example 1 and Comparative Example 3.

Detailed ways

The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only used to help understand the present invention and should not be regarded as specific limitations of the present invention.

The preparation method of the silk fibroin used in the embodiment of the present invention is as follows: 40g of natural silkworm silk is placed in 2L of sodium carbonate solution with a concentration of 0.05% and boiled for 30min, repeated three times, and then fully washed with deionized water and dried to obtain silkworm silk fiber. Then the silk fiber is dissolved in a 9.3mol/L lithium bromide (LiBr) solution with a bath ratio of 1:10, and stirred at 60°C for 1h to obtain a silk fibroin mixed solution. After the mixed solution is cooled, the mixed solution is loaded into a dialysis bag (molecular weight cutoff 8-12kD), dialyzed with deionized water for 5 days, changing the water every 12h, and filtered after the dialysis to obtain a silk fibroin solution. The silk fibroin solution is placed in a -80°C refrigerator, and then freeze-dried to obtain silk fibroin.

Example 1

This embodiment provides a silk fibroin-based three-dimensional structure double-layer membrane and a preparation method thereof, the preparation method comprising the following steps:

(1) The preparation method of the porous loose membrane is as follows: the freeze-dried silk fibroin and nano-hydroxyapatite particles are dissolved in 10 mL of hexafluoroisopropanol (HFIP) at a mass ratio of 5:1 to prepare a mixed solution with a mass volume ratio of 15%, and the mixture is dispersed by ultrasonic oscillation for 30 minutes. After stirring with a magnetic stirrer for 24 hours at room temperature, a milky SF/nHA electrospinning solution is obtained. The SF/nHA electrospinning solution is transferred to a 10 mL medical syringe in batches, and a 20G needle is connected to the syringe. The syringe is then placed in a propulsion pump, connected to a high-voltage generator, the voltage is set to 15 kV, the propulsion pump rate is set to 1.0 mL/h, and an electrospinning drum is placed under the propulsion pump to receive the fiber, the rotation speed is 1000 r/min, and the receiving distance is fixed to 12 cm. The whole process is carried out at room temperature, the air humidity is 15-20%, and the prepared single-layer membrane is named nHA@SF.

(2) The preparation method of the dense membrane is as follows: silk fibroin is dissolved in hexafluoroisopropanol to prepare a mixed solution with a mass volume ratio of 15%. After stirring with a magnetic stirrer for 12 hours at room temperature, a clear and transparent SF electrospinning solution is obtained. The SF electrospinning solution is transferred to a 10mL medical syringe, a 20G needle is connected to the syringe, and then the syringe is placed in a propulsion pump, connected to a high-voltage generator, the voltage is set to 15kV, the propulsion pump rate is set to 1.2mL/h, and an electrospinning drum is placed under the propulsion pump to receive the fiber, the speed is 1000r/min, and the receiving distance is fixed to 15cm. The whole process is carried out at room temperature and the air humidity is 15-20%. The single-layer membrane prepared in this step is named 10SF.

(3) The preparation method of the nanofiber double-layer membrane based on silk fibroin is as follows: on the basis of the porous loose membrane formed by the SF/nHA layer, the SF/PLCL layer of dense membrane is spun to form a silk-based three-dimensional structure double-layer membrane. The prepared double-layer membrane is named nHA@SF-10SF. After spinning, the electrospun membrane is placed in a vacuum drying oven for 48 hours to remove the residual organic solvent on the membrane, and then cross-linked in an EDC-NHS cross-linker solution (EDC: NHS mass ratio is 2:1 dissolved in anhydrous ethanol, and the mass volume ratio of EDC-NHS to anhydrous ethanol is 3:10) for 4 hours, and the residual cross-linker is rinsed with deionized water and placed in a freeze dryer to finally dry the silk-based three-dimensional structure double-layer membrane.

Example 2

This embodiment provides a silk fibroin-based three-dimensional structure double-layer membrane and a preparation method thereof, the preparation method comprising the following steps:

(1) The preparation method of the porous loose membrane is as follows: freeze-dried silk fibroin and nano-hydroxyapatite particles are dissolved in 10 mL of hexafluoroisopropanol (HFIP) at a mass ratio of 5:1 to prepare a mixed solution with a mass volume ratio of 15%. Ultrasonic oscillation treatment is performed for 30 minutes to disperse them, and a milky SF/nHA electrospinning solution is obtained after stirring with a magnetic stirrer for 24 hours at room temperature. The SF/nHA electrospinning solution is transferred into a 10 mL medical syringe in batches, and a 20G needle is connected to the syringe. The syringe is then placed in a propulsion pump, connected to a high-voltage generator, the voltage is set to 15 kV, the propulsion pump rate is set to 1.0 mL/h, and an electrospinning drum is placed under the propulsion pump to receive the fiber, the rotation speed is 1000 r/min, and the receiving distance is fixed to 12 cm. The entire process is carried out at room temperature, the air humidity is 15-20%, and the prepared single-layer membrane is named nHA@SF.

(2) The preparation method of the dense membrane is as follows: silk fibroin and poly (L-lactide-caprolactone) are dissolved in hexafluoroisopropanol in a mass ratio of 8:2, respectively, to prepare a mixed solution with a mass volume ratio of 15%. After stirring with a magnetic stirrer for 12 hours at room temperature, a clear and transparent SF/PLCL electrospinning solution is obtained. The SF/PLCL electrospinning solution is transferred to a 10mL medical syringe, a 20G needle is connected to the syringe, and then the syringe is placed in a propulsion pump, connected to a high-voltage generator, the voltage is set to 15kV, the propulsion pump rate is set to 1.2mL/h, and an electrospinning drum is placed under the propulsion pump to receive the fiber, the rotation speed is 1000r/min, and the receiving distance is fixed to 15cm. The whole process is carried out at room temperature and the air humidity is 15-20%. The single-layer membrane prepared in this step is named 8SF/2P.

(3) The preparation method of the nanofiber double-layer membrane based on silk fibroin is as follows: on the basis of the porous loose membrane formed by the SF/nHA layer, the SF/PLCL layer of dense membrane is spun to form a silk-based three-dimensional structure double-layer membrane. The prepared double-layer membranes are named nHA@SF-8SF/2P. After spinning, the electrospun membrane is placed in a vacuum drying oven for 48 hours to remove the residual organic solvent on the membrane, and then cross-linked in an EDC-NHS cross-linker solution (EDC: NHS mass ratio is 2:1 dissolved in anhydrous ethanol, and the mass volume ratio of EDC-NHS to anhydrous ethanol is 3:10) for 4 hours, the solvent is anhydrous ethanol, the residual cross-linker is rinsed with deionized water and placed in a freeze dryer, and finally the silk-based three-dimensional structure double-layer membrane is dried.

Example 3

This embodiment provides a silk fibroin-based three-dimensional structure double-layer membrane and a preparation method thereof, the preparation method comprising the following steps:

(1) The preparation method of the porous loose membrane is as follows: freeze-dried silk fibroin and nano-hydroxyapatite particles are dissolved in 10 mL of hexafluoroisopropanol (HFIP) at a mass ratio of 5:1 to prepare a mixed solution with a mass volume ratio of 15%. Ultrasonic oscillation treatment is performed for 30 minutes to disperse them, and a milky SF/nHA electrospinning solution is obtained after stirring with a magnetic stirrer for 24 hours at room temperature. The SF/nHA electrospinning solution is transferred into a 10 mL medical syringe in batches, and a 20G needle is connected to the syringe. The syringe is then placed in a propulsion pump, connected to a high-voltage generator, the voltage is set to 15 kV, the propulsion pump rate is set to 1.0 mL/h, and an electrospinning drum is placed under the propulsion pump to receive the fiber, the rotation speed is 3000 r/min, and the receiving distance is fixed to 12 cm. The whole process is carried out at room temperature, the air humidity is 15-20%, and the prepared single-layer membrane is named nHA@SF.

(2) The preparation method of the dense membrane is as follows: silk fibroin and poly (L-lactide-caprolactone) are dissolved in hexafluoroisopropanol at a mass ratio of 5:5, respectively, to prepare a mixed solution with a mass volume ratio of 15%. After stirring with a magnetic stirrer for 12 hours at room temperature, a clear and transparent SF/PLCL electrospinning solution is obtained. The SF/PLCL electrospinning solution is transferred to a 10mL medical syringe, a 20G needle is connected to the syringe, and then the syringe is placed in a propulsion pump, connected to a high-voltage generator, the voltage is set to 15kV, the propulsion pump rate is set to 1.2mL/h, and an electrospinning drum is placed under the propulsion pump to receive the fiber, the rotation speed is 3000r/min, and the receiving distance is fixed to 15cm. The whole process is carried out at room temperature and the air humidity is 15-20%. The single-layer membrane prepared in this step is named 5SF/5P.

(3) The preparation method of the nanofiber double-layer membrane based on silk fibroin is as follows: on the basis of the porous loose membrane formed by the SF/nHA layer, a dense membrane of the SF/PLCL layer is spun to form a silk-based three-dimensional structure double-layer membrane. The prepared double-layer membranes are named nHA@SF-5SF/5P. After spinning, the electrospun membrane is placed in a vacuum drying oven for 48 hours to remove the residual organic solvent on the membrane, and then cross-linked in an EDC-NHS cross-linker solution (EDC: NHS mass ratio is 2:1 dissolved in anhydrous ethanol, and the mass volume ratio of EDC-NHS to anhydrous ethanol is 3:10) for 4 hours, the solvent is anhydrous ethanol, the residual cross-linker is rinsed with deionized water and placed in a freeze dryer, and finally the silk-based three-dimensional structure double-layer membrane is dried.

The schematic diagram of the structure of the silk-based three-dimensional structure double-layer membrane prepared by the present invention is shown in Figure 1A, the scanning electron microscope (Hitachi, TM-1000) image of the outer layer 5SF/5P nanofiber membrane prepared in this embodiment is shown in Figure 1B, the scanning electron microscope image of the inner layer nHA@SF nanofiber membrane is shown in Figure 1C, and the scanning electron microscope image of the silk-based three-dimensional structure double-layer membrane is shown in Figure 1D. It can be seen from the figure that protruding bulges or nodules are observed on the fibers containing hydroxyapatite on the surface of the nHA@SF nanofiber membrane, and the porosity of the nanofiber membrane doped with hydroxyapatite is higher than that of the undoped nanofiber membrane. The side-section results show that nHA@SF is relatively loose, while 5SF/5P is relatively dense, and the bonding between the two layers is relatively tight.

The thickness of the nanofiber membrane was measured and averaged, and the obtained data is shown in the following Table 1. While achieving a double-layer structure, it still has a small thickness, and it is expected to be able to better fit the periodontal tissue during clinical use.

Table 1

Example 1 0.1093mm Example 2 0.0982mm Example 3 0.9734mm

The prepared membranes were characterized by infrared (Thermo Fisher, NicoletiN 10). Figure 2 shows the infrared spectrum of the nanofiber membranes, including (A) inner layer nHA@SF nanofiber membrane, (B) outer layer 5SF/5P nanofiber membrane; it can be seen from the figure that the chemical composition of the silk-based nanofiber double-layer membrane can be verified according to the position and intensity of the absorption peaks. Specifically, all nanofiber membranes containing silk (SF) show obvious absorption peaks at 1623cm ^-1 , 1518cm ^-1 and 1233cm ^-1 , corresponding to Silk-I and Silk-II (NH) and Silk-III (CN) in SF, respectively. After loading nanohydroxyapatite (nHA), nHA/SF and PLCL/SF/nHA composite membranes show absorption peaks at 1045cm ^-1 , indicating the presence of PO bonds in the membranes, while the absorption peak at 601cm ^-1 is derived from the OPO phosphate group in nHA. These results confirm that nHA is successfully loaded into the SF/PLCL/nHA composite membrane. In addition, a strong absorption peak was observed at 1752cm ^-1 , corresponding to the typical asymmetric stretching of the carbonyl group of polylactic acid (PLA). The absorption peaks at 2995cm ^-1 and 2945cm ^-1 belong to the asymmetric and symmetric CH stretching vibrations of PLA, respectively. The absorption peaks at 1454cm ^-1 and 1382cm ^-1 belong to the symmetric and asymmetric bending CH deformation of PLA. The peaks at 1181cm ^-1 , 1130cm ^-1 and 1085cm ^-1 are due to the CO stretching of PLA. In summary, the FTIR spectrum analysis can conclude that the prepared silk-based nanofiber double-layer membrane has the basic characteristic groups of the constituent substances and has successfully loaded nHA.

The prepared membrane was characterized by X-ray diffraction (Rigaku, D/max-2550PC), and the results are shown in Figure 3. It can be seen from the figure that two main characteristic diffraction peaks appeared in the XRD spectrum of pure PLCL material, located at 2θ=16.7° and 19.0°, respectively, which indicates that there is a typical semi-crystalline structure in PLCL. This is because the solvent used for PLCL is HFIP, which is a benign solvent for PLCL. In dilute solution, the PLCL molecular chain can be completely surrounded by HFIP molecules, and the molecular chains are independent of each other. The polymer exists in the solution in the form of isolated coils, which reduces the contact between polymers. The molecular chains are fully extended to avoid mutual entanglement, and these extended polymer chains tend to be arranged in an orderly manner. However, the fiber membrane doped with SF and nHA did not show sharp peaks, indicating that these composite materials have no obvious crystalline structure. Most of PLCL and SF exist in the fiber membrane as amorphous structures, and only a small part of the ordered structure is displayed in the form of broad peaks. After PLCL and SF are compounded, the ordered structure of some polymer chains is disrupted.

The contact angle of the prepared membrane was tested using a contact angle meter (Kruss, DSA-30). The results are shown in Figure 4. It can be seen that the contact angles of 10SF, 8SF/2P, 5SF/5P and nHA@SF are 74.76°±3.22°, 98.76°±1.72°, 108.69°±2.49°, and 67.72°±2.29°, respectively. The experimental results show that as the proportion of SF content decreases, the hydrophobicity of the silk-based nanofiber double-layer membrane increases. Because the hydroxyl groups in SF can form hydrogen bonds with water molecules, the membrane surface has a more significant hydrophilicity. At the same time, nHA@SF has a large surface roughness due to the addition of nHA, and the parameters set when preparing nHA@SF by electrospinning make the membrane itself have a large porosity, so it is the most hydrophilic. The film materials have different roughness and surface area, and the surface tension generated will be different, which will affect the contact angle. Generally, when the contact angle is less than 90°, the rough surface has a larger effective area and greater surface tension, making it easier to wet. The experimental results show that the contact angles of 10SF and nHA@SF are less than 90°, and the contact angles of 8SF/2P and 5SF/5P are also close to 90°. Therefore, both the inner and outer layers of the double-layer membrane prepared in this experiment have good hydrophilicity, which is helpful to a certain extent for the adhesion, growth and reproduction of cells on the membrane surface.

The mechanical properties of the silk-based three-dimensional structure double-layer membrane obtained in Example 1-3 were tested using a computerized tensile and pressure testing machine (Shanghai Hengyu, HY-940FS), as shown in Figure 5. Among them, (A) stress-strain curve, (B) breaking strength, (C) elongation at break, (D) Young's modulus; the addition of an appropriate amount of PLCL can significantly enhance the fracture toughness of the fiber membrane. The maximum tensile strength value of the nHA@SF-10SF group without PLCL is 2.576MPa, while the maximum tensile strength of the nHA@SF-5SF/5P with a ratio of SF to PLCL of 5:5 is 1.455MPa, and PLCL weakens the tensile strength to a certain extent. It can be concluded from the experimental results that the addition of PLCL in an appropriate amount can improve the mechanical properties of the silk nanofiber composite membrane, while the addition of too much PLCL will have a certain negative impact on its mechanical properties. Therefore, for the preparation of silk nanofiber composite membranes, it is necessary to properly control the amount of PLCL added while ensuring the mechanical properties.

Example 4

This embodiment provides a silk fibroin-based three-dimensional structure double-layer membrane and a preparation method thereof, the preparation method comprising the following steps:

(1) The preparation method of the porous loose membrane is as follows: the freeze-dried silk fibroin and nano-hydroxyapatite particles are dissolved in 10 mL of hexafluoroisopropanol (HFIP) at a mass ratio of 4:1 to prepare a mixed solution with a mass volume ratio of 15%. The mixture is dispersed by ultrasonic oscillation for 30 minutes, and a milky SF/nHA electrospinning solution is obtained after stirring with a magnetic stirrer for 24 hours at room temperature. The SF/nHA electrospinning solution is transferred into a 10 mL medical syringe in batches, and a 20G needle is connected to the syringe. The syringe is then placed in a propulsion pump, connected to a high-voltage generator, the voltage is set to 15 kV, the propulsion pump rate is set to 1.0 mL/h, and an electrospinning drum is placed under the propulsion pump to receive the fiber, the rotation speed is 1000 r/min, and the receiving distance is fixed to 12 cm. The whole process is carried out at room temperature, the air humidity is 15-20%, and the prepared single-layer membrane is named nHA@SF.

(2) The preparation method of the dense membrane is as follows: silk fibroin and poly (L-lactide-caprolactone) are dissolved in hexafluoroisopropanol in a mass ratio of 8:2, respectively, to prepare a mixed solution with a mass volume ratio of 15%. After stirring with a magnetic stirrer for 12 hours at room temperature, a clear and transparent SF/PLCL electrospinning solution is obtained. The SF/PLCL electrospinning solution is transferred to a 10mL medical syringe, a 20G needle is connected to the syringe, and then the syringe is placed in a propulsion pump, connected to a high-voltage generator, the voltage is set to 15kV, the propulsion pump rate is set to 1.2mL/h, and an electrospinning drum is placed under the propulsion pump to receive the fiber, the rotation speed is 1000r/min, and the receiving distance is fixed to 15cm. The whole process is carried out at room temperature and the air humidity is 15-20%. The single-layer membrane prepared in this step is named 8SF/2P.

(3) The preparation method of the nanofiber double-layer membrane based on silk fibroin is as follows: on the basis of the porous loose membrane formed by the SF/nHA layer, a dense membrane of the SF/PLCL layer is spun to form a silk-based three-dimensional structure double-layer membrane. The prepared double-layer membranes are named nHA@SF-8SF/2P. After spinning, the electrospun membrane is placed in a vacuum drying oven for 48 hours to remove the residual organic solvent on the membrane, and then cross-linked in the EDC-NHS system for 4 hours, the solvent is anhydrous ethanol, and the residual cross-linking agent is rinsed with deionized water and placed in a freeze dryer to finally dry the silk-based three-dimensional structure double-layer membrane.

Example 5

This embodiment provides a silk fibroin-based three-dimensional structure double-layer membrane and a preparation method thereof, the preparation method comprising the following steps:

(1) The preparation method of the porous loose membrane is as follows: freeze-dried silk fibroin and nano-hydroxyapatite particles are dissolved in 10 mL of hexafluoroisopropanol (HFIP) at a mass ratio of 20:3 to prepare a mixed solution with a mass volume ratio of 15%. Ultrasonic oscillation treatment is performed for 30 minutes to disperse them, and a milky SF/nHA electrospinning solution is obtained after stirring with a magnetic stirrer for 24 hours at room temperature. The SF/nHA electrospinning solution is transferred into a 10 mL medical syringe in batches, and a 20G needle is connected to the syringe. The syringe is then placed in a propulsion pump, connected to a high-voltage generator, the voltage is set to 15 kV, the propulsion pump rate is set to 1.0 mL/h, and an electrospinning drum is placed under the propulsion pump to receive the fiber, the rotation speed is 1000 r/min, and the receiving distance is fixed to 12 cm. The entire process is carried out at room temperature, the air humidity is 15-20%, and the prepared single-layer membrane is named nHA@SF.

(2) The preparation method of the dense membrane is as follows: silk fibroin and poly (L-lactide-caprolactone) are dissolved in hexafluoroisopropanol in a mass ratio of 8:2, respectively, to prepare a mixed solution with a mass volume ratio of 15%. After stirring with a magnetic stirrer for 12 hours at room temperature, a clear and transparent SF/PLCL electrospinning solution is obtained. The SF/PLCL electrospinning solution is transferred to a 10mL medical syringe, a 20G needle is connected to the syringe, and then the syringe is placed in a propulsion pump, connected to a high-voltage generator, the voltage is set to 15kV, the propulsion pump rate is set to 1.2mL/h, and an electrospinning drum is placed under the propulsion pump to receive the fiber, the rotation speed is 1000r/min, and the receiving distance is fixed to 15cm. The whole process is carried out at room temperature and the air humidity is 15-20%. The single-layer membrane prepared in this step is named 8SF/2P.

(3) The preparation method of the nanofiber double-layer membrane based on silk fibroin is as follows: on the basis of the porous loose membrane formed by the SF/nHA layer, a dense membrane of the SF/PLCL layer is spun to form a silk-based three-dimensional structure double-layer membrane. The prepared double-layer membranes are named nHA@SF-8SF/2P. After spinning, the electrospun membrane is placed in a vacuum drying oven for 48 hours to remove the residual organic solvent on the membrane, and then cross-linked in the EDC-NHS system for 4 hours, the solvent is anhydrous ethanol, and the residual cross-linking agent is rinsed with deionized water and placed in a freeze dryer to finally dry the silk-based three-dimensional structure double-layer membrane.

The scanning electron microscope (Hitachi, TM-1000) images of the inner nanofiber membranes prepared in Example 3, Example 4, and Example 5 are shown in Figure 6 A, B, and C, respectively. It is observed that nHA is doped between the fibers. The nHA content in Example 4 is the highest, but the fibers are thinner than those in Example 3. It is speculated that the excessive nHA content blocks the electrospinning needle, making the fiber diameter smaller; and the nHA content in Example 5 is too low compared to Example 3, making nHA unevenly distributed in the fibers. In summary, the ratio of SF to nHA in Example 3 is the most appropriate.

Comparative Example 1

The only difference from Example 1 is that the preparation method does not include step (3), that is, no cross-linking occurs.

Comparative Example 2

The only difference from Example 1 is that the solvent hexafluoroisopropanol used in step (1) and step (2) is replaced by trifluoroacetic acid.

Comparative Example 3

The only difference from Example 1 is that the cross-linking agent EDC-NHS system in step (3) is replaced by tannic acid.

The membranes obtained in Example 2 and Comparative Example 1 were subjected to an in vitro degradation test, and the test method was to use artificial saliva (pH = 6.8) to simulate the degradation process of the silk-based nanofiber double-layer membrane. The cross-linked nHA@SF-8SF/2P and uncross-linked nHA@SF-8SF/2P samples were cut into 2.5 cm × 2.5 cm sizes, and their masses were recorded as W ₀ after drying and weighing. Subsequently, the samples were transferred to 5 mL of artificial saliva and placed on a 37°C shaker. At 3, 7, 14, 21, 28 and 49 days, after taking out the samples, they were rewashed several times with deionized water and freeze-dried, and the mass was recorded as W. The remaining mass percentage (Remaining Mass) of the sample was calculated by the formula: Remaining Mass (%) = (W/W ₀ ) × 100%.

The test results are shown in Figure 7. It can be seen that the cross-linked nHA@SF-8SF/2P double-layer membrane presents a relatively stable degradation curve as a whole. After 7 weeks of degradation, the mass loss of the double-layer membrane remains above 75%. The uncross-linked nHA@SF-8SF/2P double-layer membrane is basically completely degraded in the first week. This shows that the use of cross-linking agents has a great effect on the degradation performance of the material.

The membranes obtained in Example 2 and Comparative Example 2 were observed by SEM. The test method was as follows: 8SF/2P samples whose electrospinning solvent was hexafluoroisopropanol and 8SF/2P samples whose solvent was trifluoroacetic acid were cut into squares of 1.0 cm×1.0 cm. Then, a suitable SEM sample stage was selected, and the cut samples were fixed on the sample stage with conductive glue. The samples were sprayed with gold for 60 seconds at a DC current of 4 mA under a certain vacuum degree, so that the sample surface had conductivity. Finally, the samples were placed under a SEM for photographing.

The test results are shown in Figure 8. (A) The solvent is hexafluoroisopropanol, and (B) The solvent is trifluoroacetic acid. It can be seen that when the electrospinning solvent is hexafluoroisopropanol, a relatively continuous fiber structure with uniform thickness and size can be formed. When the electrospinning solvent is trifluoroacetic acid, a complete fiber structure cannot be formed and droplets fall.

The films obtained in Example 1 and Comparative Example 3 were observed.

The test results are shown in Figure 9. (A) The crosslinking agent is tannic acid, and (B) the crosslinking agent is EDC-NHS. It can be seen that the surface of the membrane with tannic acid as the crosslinking agent is relatively rough after drying, and is easily broken after contact; the surface of the membrane with EDC-NHS as the crosslinking agent is relatively smooth, and is not easy to break after multiple folding and bending.

The applicant declares that the present invention uses the above-mentioned embodiments to illustrate the silk-based three-dimensional structured double-layer membrane for periodontal regeneration and its preparation method and application, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims (26)

1. A silk-based three-dimensional double-layer membrane for periodontal regeneration, characterized in that it comprises an inner porous loose layer and an outer dense layer, wherein the porous loose layer is a nanofiber membrane composed of silk fibroin and nano-hydroxyapatite, and the dense layer is a nanofiber membrane composed of silk fibroin and biodegradable materials, and a cross-linked structure is formed between the porous loose layer and the dense layer; The silk-based three-dimensional structured double-layer membrane for periodontal regeneration is prepared by the following method, which comprises the following steps: (1) Using a solution containing silk fibroin and nano-hydroxyapatite as an electrospinning solution, a porous loose layer is obtained by electrospinning; (2) using a solution containing silk fibroin and biodegradable materials as an electrospinning solution, and receiving and obtaining a dense layer on the obtained porous loose layer through electrospinning; (3) Using a cross-linking agent to cross-link the porous loose layer and the dense layer to obtain the silk-based three-dimensional structured double-layer membrane for periodontal regeneration. 2. The silk-based three-dimensional structured double-layer membrane for periodontal regeneration according to claim 1, characterized in that the mass ratio of nano-hydroxyapatite in the porous loose layer is 10% to 30%. 3 . The silk-based three-dimensional structured double-layer membrane for periodontal regeneration according to claim 1 , wherein the particle size of the nano-hydroxyapatite is 20 to 40 nm. 4. The silk-based three-dimensional structured double-layer membrane for periodontal regeneration according to claim 1, characterized in that the weight percentage of silk fibroin in the dense layer is 50% to 100%. 5. The silk-based three-dimensional structure double-layer membrane for periodontal regeneration according to claim 1, characterized in that the biodegradable material is selected from any one of polylactic acid, polycaprolactone or polyhydroxybutyrate-valeric acid copolyester or a combination of at least two thereof. 6 . The silk-based three-dimensional structured double-layer membrane for periodontal regeneration according to claim 1 , wherein the cross-linking agent used for the cross-linking is carbodiimide salt and hydroxysuccinimide. 7 . The silk-based three-dimensional structured double-layer membrane for periodontal regeneration according to claim 6 , wherein the mass ratio of the carbodiimide salt to hydroxysuccinimide is 20% to 30%. 8. The method for preparing a silk fibroin-based three-dimensional structured double-layer membrane for periodontal regeneration according to any one of claims 1 to 7, characterized in that the preparation method comprises the following steps: (1) Using a solution containing silk fibroin and nano-hydroxyapatite as an electrospinning solution, a porous loose layer is obtained by electrospinning; (2) using a solution containing silk fibroin and biodegradable materials as an electrospinning solution, and receiving and obtaining a dense layer on the obtained porous loose layer through electrospinning; (3) Using a cross-linking agent to cross-link the porous loose layer and the dense layer to obtain the silk-based three-dimensional structured double-layer membrane for periodontal regeneration. 9. The preparation method according to claim 8, characterized in that the total mass percentage of silk fibroin and nano-hydroxyapatite in the solution containing silk fibroin and nano-hydroxyapatite in step (1) is 15% to 25%. 10 . The preparation method according to claim 8 , characterized in that the solvent of the solution containing silk fibroin and nano-hydroxyapatite in step (1) is hexafluoroisopropanol. 11. The preparation method according to claim 8, characterized in that the preparation process of the solution containing silk fibroin and nano-hydroxyapatite in step (1) is: adding freeze-dried silk fibroin and nano-hydroxyapatite particles to hexafluoroisopropanol to obtain a mixed solution, treating the mixed solution with ultrasonic vibration, and then stirring to obtain the solution containing silk fibroin and nano-hydroxyapatite. 12. The preparation method according to claim 11, characterized in that the ultrasonic frequency during the ultrasonic oscillation treatment is 19-23 KHz, and the treatment time is 10-40 min. 13. The preparation method according to claim 11, characterized in that the stirring time is 3-48 hours. 14. The preparation method according to claim 8, characterized in that, during the electrospinning in step (1), the voltage is set to 10-20 kV, the sample feed rate is set to 0.8-1.5 mL/h, the fiber is received by a roller, the roller speed is 800-3000 r/min, and the receiving distance is 10-15 cm; The electrospinning in step (1) is carried out at room temperature with an air humidity of 15-20%. 15. The preparation method according to claim 8, characterized in that the mass percentage of silk fibroin and biodegradable material in the solution containing silk fibroin and biodegradable material in step (2) is 10% to 20%. 16. The preparation method according to claim 8, characterized in that the solvent of the solution containing silk fibroin and biodegradable material in step (2) is at least one of hexafluoroisopropanol, trifluoroacetic acid, acetic acid or tetrahydrofuran. 17. The preparation method according to claim 8, characterized in that the preparation process of the solution containing silk fibroin and biodegradable material in step (2) is: adding silk fibroin and biodegradable material to hexafluoroisopropanol to obtain a mixed solution, stirring, and obtaining the solution containing silk fibroin and biodegradable material. 18. The preparation method according to claim 17, characterized in that the stirring time is 3-24 hours. 19. The preparation method according to claim 8, characterized in that, during the electrospinning in step (2), the voltage is set to 10-20 kV, the sample feed rate is set to 0.8-1.5 mL/h, the fiber is received by a roller, the roller speed is 800-3000 r/min, and the receiving distance is 10-15 cm. 20. The preparation method according to claim 8, characterized in that the electrospinning in step (2) is carried out at room temperature with an air humidity of 15-20%. 21. The preparation method according to claim 8, characterized in that the cross-linking agent in step (3) is carbodiimide salt and hydroxysuccinimide. 22. The preparation method according to claim 21, characterized in that the mass ratio of the carbodiimide salt to hydroxysuccinimide is 20% to 30%. 23. The preparation method according to claim 8, characterized in that the cross-linking in step (3) is carried out at room temperature and the cross-linking time is 1-6 hours. 24. The preparation method according to claim 8, characterized in that the cross-linking in step (3) is carried out in an organic solvent, and the organic solvent is anhydrous ethanol. 25. The preparation method according to claim 8, characterized in that, in step (3), the double-layer membrane obtained in step (2) is dried before cross-linking to remove the organic solvent remaining on the membrane. 26. Use of the silk-based three-dimensional structured double-layer membrane for periodontal regeneration according to any one of claims 1 to 7 in the preparation of oral implant materials.