CN116392647A

CN116392647A - Silk fibroin-based three-dimensional structure bilayer membrane for periodontal regeneration and preparation method and application thereof

Info

Publication number: CN116392647A
Application number: CN202310557229.4A
Authority: CN
Inventors: 王驰巍; 仇可新
Original assignee: Shanghai Liyuan Medical Technology Co ltd
Current assignee: Shanghai Liyuan Medical Technology Co ltd
Priority date: 2023-05-17
Filing date: 2023-05-17
Publication date: 2023-07-07
Anticipated expiration: 2043-05-17
Also published as: CN116392647B

Abstract

The invention provides a silk fibroin-based three-dimensional structure double-layer membrane for periodontal regeneration, a preparation method and application thereof. The silk fibroin-based three-dimensional structure double-layer membrane has a three-dimensional structure, has a good mechanical barrier effect, can induce bone tissue regeneration effect, has a soft membrane structure, is well attached to periodontal tissues, has good mechanical properties and degradation properties, is simple to operate, has the advantage of mild experimental preparation conditions, and is suitable for mass production.

Description

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

Technical Field

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

Background

The oral implantation refers to making artificial tooth roots, dental crowns and the like by using biological materials to repair missing teeth and surrounding tissues, and realize stable and comfortable chewing function and tooth appearance for a long time. However, after the teeth are lost, the alveolar bone is gradually shrunken due to the factors such as loss of functional stimulus, so that the problem of insufficient bone mass commonly seen in implant restoration is one of the main challenges facing the field. Therefore, prior to oral implant surgery, the bone mass of the patient needs to be assessed. In the case of a patient with a small amount of alveolar bone, bone grafting is required first, and human bone is replaced with a scientific and technological regenerating material such as bone powder and periosteum, which is called guided bone tissue regeneration (Guided bone regeneration, GBR). In the whole implantation repair process, a part of patients need to perform bone increment operation, and a part of patients need to use bone powder and an oral repair film. The adoption of the oral cavity repairing film material can promote bone regeneration in dental implantation, improve the stability of the implant, ensure good development of bones and implant bones, have good treatment effect, and have important values for improving the oral health of patients and improving the life quality.

By applying the oral repair film to cover the bone defect site, a mechanical barrier can be formed, thereby achieving the sealing effect. The covering can relieve the pressure of covered tissues, and protect blood clots to form good osteogenic space. The barrier can block fibroblasts, connective tissues and the like which influence bone formation, so that precursor osteoblasts with growth potential enter a bone defect area, thereby inducing bone repair regeneration of the bone defect area and increasing the success rate of oral implantation repair.

The oral cavity repairing films are various in types, and the traditional oral cavity repairing films which are made of alloy materials and high polymer materials are gradually eliminated due to the defects of poor biocompatibility, nondegradable property and the like. Collagen-based oral repair membranes currently occupy the major market, but there are more or less some drawbacks to the same type of products already on the market:

(1) The collagen degradation speed is high, and the spatial structure cannot be maintained in the later period of repair;

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

(3) The loose surface has poor suction permeability, can not effectively lock blood permeation and has poor effect of inducing bone regeneration;

(4) The mouth repair film has stiff hand feeling and poor attaching effect, and can influence the convenience of operation.

In contrast, silk Fibroin (SF) has good biodegradability, mechanical strength and biocompatibility as a natural polymer material, and is widely used in tissue engineering of bone, cartilage, liver and skin. However, silk fibroin has poor adhesion and hydrophilicity, limiting its use in medical materials. After crosslinking modification, the silk fibroin can induce to form a beta-sheet structure, so that the cell adhesion and the elastic modulus of the material are improved. The electrostatic spinning technology can be used for preparing the nano-scale silk fibroin fiber net, and the mechanical strength of the silk fibroin fiber net is higher and is closer to the physical structure of a natural periosteum. The electrospun monolayer film has the defects of thin thickness, poor strength, easy swelling in water and the like.

Accordingly, it is desirable in the art to develop a material that can have desirable tissue conformability, mechanical properties, and degradation cycles.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a silk fibroin-based three-dimensional structure double-layer membrane for periodontal regeneration, and a preparation method and application thereof.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

in one aspect, the invention provides a silk fibroin-based three-dimensional structure double-layer membrane for periodontal regeneration, which comprises a porous loose layer and an outer compact layer, wherein the porous loose layer is a nanofiber membrane composed of silk fibroin and nano hydroxyapatite, the compact 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 compact layer.

In the invention, silk fibroin is compounded with nano-hydroxyapatite (nHA), so that the surface roughness is increased and the mechanical strength is greatly improved. In addition, the stability and mechanical strength of the nanofiber membrane can be further improved by adding the high-molecular biodegradable material. The porous loose layer and the dense layer of the outer layer form a cross-linked structure, and the silk fibroin can be induced to form a beta-sheet structure, so that the periodontal regenerated silk fibroin-based double-layer membrane has a three-dimensional structure, and has more ideal tissue laminating property, mechanical property and degradation period compared with a collagen membrane, and has a larger prospect in clinical application.

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

Preferably, the nano-hydroxyapatite has a particle size of 20 to 40nm, for example 20nm, 23nm, 25nm, 28nm, 30nm, 35nm, 38nm or 40nm. The silk fibroin-based three-dimensional structure double-layer film for periodontal regeneration is prepared by electrostatic spinning, so that the particle size of nano hydroxyapatite is maintained at 20-40 nm by grinding and sieving, and the nano hydroxyapatite is better dispersed in an electrostatic spinning solution to avoid sedimentation; meanwhile, the needle is not easy to block in the electrostatic spinning process, so that the spinning process is smoother.

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

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

Preferably, the crosslinking agents used for the crosslinking are carbodiimide (EDC) and hydroxysuccinimide (NHS).

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

In the invention, in the process of crosslinking the porous loose layer and the compact layer, the silk fibroin can be induced to form a beta-sheet structure, and the formed silk fibroin-based ultrathin double-layer film has better fitting property with periodontal tissues, and compared with the commercially available double-layer collagen film, the commercially available double-layer collagen film has stiff hand feeling and poor attaching effect.

In another aspect, the present invention provides a method for preparing a silk fibroin-based three-dimensional structure bilayer membrane for periodontal regeneration as described above, the method comprising the steps of:

(1) Taking a solution containing silk fibroin and nano hydroxyapatite as an electrostatic spinning solution, and obtaining a porous loose layer through electrostatic spinning;

(2) Taking a solution containing silk fibroin and a biodegradable material as an electrostatic spinning solution, and receiving and obtaining a compact layer on the obtained porous loose layer through electrostatic spinning;

(3) The crosslinking agent is used for crosslinking the multi-Kong Shusong layer and the compact layer, so that the silk fibroin-based three-dimensional structure double-layer film for periodontal regeneration is obtained.

In the invention, a dense layer of silk fibroin and biodegradable materials is formed on a porous loose layer through electrostatic spinning, and then the silk fibroin can be induced to form a beta-sheet structure through crosslinking, so that the formed silk fibroin-based ultrathin double-layer film has better fitting property with periodontal tissues, and compared with the commercially available double-layer collagen film, the commercially available double-layer collagen film has stiff hand feeling and poor attaching effect.

According to the invention, natural high molecular material Silk Fibroin (SF), biodegradable material and inorganic component nano hydroxyapatite (nHA) are compounded together to form a composite material, and the advantages are complementary, so that a silk fibroin-based three-dimensional structure double-layer membrane is constructed. The silk fibroin film is used as a main material, and has good biodegradability and biocompatibility; the biodegradable material has good biocompatibility and biodegradability, can be used as an auxiliary material together with nano hydroxyapatite, improves the defect of insufficient mechanical property of silk fibroin nanofiber, and simultaneously improves the surface roughness and the osteoinductive property.

According to the invention, the synthesis design of biological materials is carried out according to the material optimization design and the research thought of the bionic periosteum structure, the natural material silk fibroin and nano hydroxyapatite are compounded and then spun to prepare the porous membrane, and then the silk fibroin/poly L-lactide-caprolactone dense membrane is spun on the porous membrane and then crosslinked, so that the silk fibroin-based three-dimensional structure double-layer membrane with the bionic periosteum structure is constructed. The regeneration membrane has compact outer layer and can prevent fibroblast from growing in; the inner layer is loose, which is beneficial 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% -25%, such as 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 the silk fibroin and the nano hydroxyapatite in the step (1) is as follows: adding the freeze-dried silk fibroin and nano hydroxyapatite particles into hexafluoroisopropanol to obtain a mixed solution, carrying out ultrasonic vibration treatment on the mixed solution, and then stirring to obtain the solution containing the silk fibroin and the nano hydroxyapatite.

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

In the present invention, the stirring may be magnetic stirring as described above for a period of 3 to 48 hours.

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

In the invention, the electrostatic spinning solution in the step (1) is transferred to a 10mL medical injector in batches, a needle with the model of 20G is connected to the injector, and then the injector is placed into a propelling pump of an electrostatic spinning device.

Preferably, the electrospinning of step (1) is carried out at room temperature with an air humidity of 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% -20%, such as 10%, 13%, 15%, 18% or 20%.

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

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

In the invention, the electrostatic spinning solution in the step (2) is transferred to a 10mL medical injector in batches, a needle with the model of 20G is connected to the injector, and then the injector is placed into a propelling pump of an electrostatic spinning device.

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

Preferably, the cross-linking agent of step (3) is carbodiimide (EDC) and hydroxysuccinimide (NHS).

Preferably, the crosslinking of step (3) is carried out at room temperature for a period of time ranging from 1 to 6 hours, for example 1 hour, 2 hours, 3 hours, 4 hours, 5 hours or 6 hours.

Preferably, the crosslinking of step (3) is performed in an organic solvent, which is absolute ethanol.

Preferably, step (3) is performed by drying the bilayer film obtained in step (2) before crosslinking to remove the organic solvent remaining on the film.

After the crosslinking in the step (3), the residual crosslinking agent is washed clean by deionized water and then is dried in a freeze dryer.

In another aspect, the invention provides the use of a silk fibroin-based three-dimensional structured bilayer membrane for periodontal regeneration as described above in the preparation of an oral implant material.

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

in the invention, silk fibroin is compounded with nano-hydroxyapatite (nHA), so that the surface roughness is increased and the mechanical strength is greatly improved. In addition, the stability and mechanical strength of the nanofiber membrane can be further improved by adding the high-molecular biodegradable material. The porous loose layer and the dense layer of the outer layer form a cross-linked structure, and can induce silk fibroin to form a beta-sheet structure, so that the periodontal regenerated silk fibroin-based double-layer membrane has a three-dimensional structure, and the dense layer has a good mechanical barrier effect as an oral cavity repair membrane, the loose layer plays a role in inducing bone tissue regeneration, realizes the double-layer structure and simultaneously has the ultra-thin physical characteristic, the membrane structure is softer and better attached to periodontal tissues, the operation is more convenient in the clinical operation process, and the ultra-thin structure is realized and simultaneously has better mechanical property and degradation property. The preparation method disclosed by the invention has the advantages of simplicity in operation and mild experimental preparation conditions, is suitable for large-scale batch production, and has a good application prospect.

Drawings

FIG. 1 is a schematic structural view and a scanning electron microscope image of a silk fibroin-based three-dimensional structure double-layer film prepared in example 3, wherein (A) the structure schematic view of the silk fibroin-based three-dimensional structure double-layer film comprises a loose surface layer (wherein the small spheres represent nano hydroxyapatite) and a dense surface layer, (B) the scanning electron microscope image of an inner layer nHA@SF nanofiber film with a scale of 10 μm, (C) the scanning electron microscope image of an outer layer 5SF/5P nanofiber film with a scale of 10 μm, and (D) the scanning electron microscope image of a cross section of the nHA@SF-5SF/5P double-layer nanofiber film with a scale of 10 μm;

FIG. 2 is an infrared spectrum of the membrane prepared in example 3, (A) an infrared spectrum of a porous loose membrane nHA@SF, (B) an infrared spectrum of a dense membrane 5 SF/5P;

FIG. 3 is an X-ray diffraction pattern of the film prepared in example 3;

FIG. 4 is the contact angle test results of the films prepared in example 3;

FIG. 5 is a graph showing the results of mechanical property test of the silk fibroin-based three-dimensional structure bilayer membrane prepared in examples 1-3, (A) stress-strain curve, (B) breaking strength test result graph, (C) elongation at break test result graph, and (D) Young's modulus test result graph;

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

FIG. 7 is an in vitro degradation curve of the films prepared in example 2 and comparative example 1;

FIG. 8 is a scanning electron microscope image of the films prepared in example 2 and comparative example 2;

fig. 9 is a surface photograph of films prepared in example 1 and comparative example 3.

Detailed Description

The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.

The preparation method of the silk fibroin used in the embodiment of the invention comprises the following steps: and (3) putting 40g of natural silk into 2L of 0.05% sodium carbonate solution, boiling for 30min, repeating for three times, fully washing with deionized water, and drying to obtain the silk fibroin fiber. The silk fibroin fibers were then dissolved in a solution of 9.3mol/L lithium bromide (LiBr) at a bath ratio of 1:10, and stirred at 60℃for 1h to obtain a silk fibroin mixed solution. After the mixed solution is cooled, the mixed solution is filled into a dialysis bag (with the molecular weight cut-off of 8-12 kD), and is dialyzed by deionized water for 5 days, water is changed every 12 hours, and the silk fibroin solution is obtained after dialysis. The silk fibroin solution was placed in a-80 ℃ refrigerator, followed by freeze-drying to obtain silk fibroin.

Example 1

The embodiment provides a silk fibroin-based three-dimensional structure bilayer membrane and a preparation method thereof, wherein the preparation method comprises the following steps:

(1) The preparation method of the porous loose membrane comprises the following steps: the freeze-dried silk fibroin and nano hydroxyapatite particles are dissolved in 10mL Hexafluoroisopropanol (HFIP) according to a mass ratio of 5:1 to prepare a mixed solution with a mass-volume ratio of 15%, the mixed solution is dispersed by ultrasonic vibration treatment for 30min, and the mixed solution is stirred for 24 hours by a magnetic stirrer at room temperature to obtain a milky SF/nHA electrostatic spinning solution. The SF/nHA electrostatic spinning solution is transferred to a 10mL medical injector in batches, a needle head with the model of 20G is connected to the injector, then the injector is placed in a propulsion pump, a high-voltage generator is connected, the voltage is set to be 15kV, the speed of the propulsion pump is set to be 1.0mL/h, an electrostatic spinning roller is placed below the propulsion pump to receive fibers, the rotating speed is 1000r/min, and the receiving distance is fixed to be 12cm. The whole process is carried out at room temperature with the air humidity of 15-20%, and the prepared monolayer film is named nHA@SF.

(2) The preparation method of the compact film comprises the following steps: silk fibroin is dissolved in hexafluoroisopropanol to prepare a mixed solution with the mass-volume ratio of 15%. After stirring for 12 hours at room temperature by a magnetic stirrer, a clear and transparent SF electrostatic spinning solution is obtained. The SF electrostatic spinning solution is transferred to a 10mL medical injector, the injector is connected with a needle head with the model of 20G, then the injector is placed in a propulsion pump, a high-voltage generator is connected, the voltage is set to be 15kV, the speed of the propulsion pump is set to be 1.2mL/h, an electrostatic spinning roller is placed below the propulsion pump to receive fibers, the rotating speed is 1000r/min, and the receiving distance is fixed to be 15cm. The whole process is carried out at room temperature with the air humidity of 15-20%. The monolayer film prepared in this step was designated 10SF.

(3) The preparation method of the silk fibroin-based nanofiber double-layer membrane comprises the following steps: spinning the SF/PLCL layer compact film on the basis of the porous loose film formed by the SF/nHA layer to form the silk fibroin-based three-dimensional structure double-layer film. The prepared bilayer membrane is named nHA@SF-10SF. After spinning, placing the electrospun membrane in a vacuum drying oven for 48 hours to remove the residual organic solvent on the membrane, then, dissolving the electrospun membrane in EDC-NHS cross-linking agent solution (EDC: NHS mass ratio is 2:1 and is dissolved in absolute ethyl alcohol, and EDC-NHS mass volume ratio and absolute ethyl alcohol mass volume ratio is 3:10) for 4 hours, washing the residual cross-linking agent with deionized water, and then, placing the washed membrane in a freeze dryer to obtain the final dry silk fibroin-based three-dimensional structure double-layer membrane.

Example 2

(1) The preparation method of the porous loose membrane comprises the following steps: the freeze-dried silk fibroin and nano hydroxyapatite particles were dissolved in 10mL Hexafluoroisopropanol (HFIP) at a mass ratio of 5:1 to prepare a mixed solution with a mass-volume ratio of 15%. Dispersing by ultrasonic oscillation for 30min, and stirring for 24 hours by a magnetic stirrer at room temperature to obtain milky SF/nHA electrostatic spinning solution. The SF/nHA electrostatic spinning solution is transferred to a 10mL medical injector in batches, a needle head with the model of 20G is connected to the injector, then the injector is placed in a propulsion pump, a high-voltage generator is connected, the voltage is set to be 15kV, the speed of the propulsion pump is set to be 1.0mL/h, an electrostatic spinning roller is placed below the propulsion pump to receive fibers, the rotating speed is 1000r/min, and the receiving distance is fixed to be 12cm. The whole process is carried out at room temperature with the air humidity of 15-20%, and the prepared monolayer film is named nHA@SF.

(2) The preparation method of the compact film comprises the following steps: respectively taking silk fibroin and poly L-lactide-caprolactone, and respectively dissolving in hexafluoroisopropanol according to the mass ratio of 8:2 to prepare a mixed solution with the mass-volume ratio of 15%. After stirring with a magnetic stirrer for 12 hours at room temperature, a clear and transparent SF/PLCL electrostatic spinning solution is obtained. The SF/PLCL electrostatic spinning solution is transferred to a 10mL medical injector, the injector is connected with a needle head with the model of 20G, then the injector is placed in a propulsion pump, a high-voltage generator is connected, the voltage is set to be 15kV, the speed of the propulsion pump is set to be 1.2mL/h, an electrostatic spinning roller is placed below the propulsion pump to receive fibers, the rotating speed is 1000r/min, and the receiving distance is fixed to be 15cm. The whole process is carried out at room temperature, and the air humidity is 15-20%. The monolayer film prepared in this step was designated 8SF/2P.

(3) The preparation method of the silk fibroin-based nanofiber double-layer membrane comprises the following steps: spinning the SF/PLCL layer compact film on the basis of the porous loose film formed by the SF/nHA layer to form the silk fibroin-based three-dimensional structure double-layer film. The prepared double-layer films are named nHA@SF-8SF/2P respectively. After spinning, placing the electrospun membrane in a vacuum drying oven for 48 hours to remove the residual organic solvent on the membrane, then dissolving the electrospun membrane in EDC-NHS cross-linking agent solution (EDC: NHS mass ratio is 2:1 and is dissolved in absolute ethyl alcohol, EDC-NHS mass volume ratio is 3:10) for 4 hours, washing the residual cross-linking agent with deionized water, and placing the dried membrane in a freeze dryer to obtain the final silk fibroin-based three-dimensional structure double-layer membrane.

Example 3

(1) The preparation method of the porous loose membrane comprises the following steps: the freeze-dried silk fibroin and nano hydroxyapatite particles were dissolved in 10mL Hexafluoroisopropanol (HFIP) at a mass ratio of 5:1 to prepare a mixed solution with a mass-volume ratio of 15%. Dispersing by ultrasonic oscillation for 30min, and stirring for 24 hours by a magnetic stirrer at room temperature to obtain milky SF/nHA electrostatic spinning solution. And transferring the SF/nHA electrostatic spinning solution to a 10mL medical injector in batches, connecting the injector with a needle head with the model size of 20G, then placing the injector into a propulsion pump, connecting a high-voltage generator, setting the voltage to be 15kV, setting the speed of the propulsion pump to be 1.0mL/h, placing an electrostatic spinning roller below the propulsion pump to receive fibers, wherein the rotating speed is 3000r/min, and the receiving distance is fixed to be 12cm. The whole process is carried out at room temperature with the air humidity of 15-20%, and the prepared monolayer film is named nHA@SF.

(2) The preparation method of the compact film comprises the following steps: respectively taking silk fibroin and poly L-lactide-caprolactone, and respectively dissolving in hexafluoroisopropanol according to the mass ratio of 5:5 to prepare a mixed solution with the mass-volume ratio of 15%. After stirring with a magnetic stirrer for 12 hours at room temperature, a clear and transparent SF/PLCL electrostatic spinning solution is obtained. The SF/PLCL electrostatic spinning solution is transferred to a 10mL medical injector, the injector is connected with a needle head with the model dimension of 20G, then the injector is placed in a propulsion pump, a high-voltage generator is connected, the voltage is set to be 15kV, the speed of the propulsion pump is set to be 1.2mL/h, an electrostatic spinning roller is placed below the propulsion pump to receive fibers, the rotating speed is 3000r/min, and the receiving distance is fixed to be 15cm. The whole process is carried out at room temperature, and the air humidity is 15-20%. The monolayer film prepared in this step was designated 5SF/5P.

(3) The preparation method of the silk fibroin-based nanofiber double-layer membrane comprises the following steps: spinning the SF/PLCL layer compact film on the basis of the porous loose film formed by the SF/nHA layer to form the silk fibroin-based three-dimensional structure double-layer film. The prepared double-layer films are named nHA@SF-5SF/5P respectively. After spinning, placing the electrospun membrane in a vacuum drying oven for 48 hours to remove the residual organic solvent on the membrane, then dissolving the electrospun membrane in EDC-NHS cross-linking agent solution (EDC: NHS mass ratio is 2:1 and is dissolved in absolute ethyl alcohol, EDC-NHS mass volume ratio is 3:10) for 4 hours, washing the residual cross-linking agent with deionized water, and placing the dried membrane in a freeze dryer to obtain the final silk fibroin-based three-dimensional structure double-layer membrane.

The structure schematic diagram of the silk fibroin-based three-dimensional structure double-layer film prepared by the invention is shown in a graph A in fig. 1, the scanning electron microscope (Hitachi, TM-1000) graph of the outer layer 5SF/5P nanofiber film prepared by the embodiment is shown in a graph B in fig. 1, the scanning electron microscope graph of the inner layer nHA@SF nanofiber film is shown in a graph C in fig. 1, the scanning electron microscope graph of the silk fibroin-based three-dimensional structure double-layer film is shown in a graph D in fig. 1, and as can be seen from the graph, protruding bulges or nodules are observed on the fiber containing hydroxyapatite on the surface of the nHA@SF nanofiber film, and the porosity of the nanofiber film doped with the hydroxyapatite is higher than that of the undoped nanofiber film. The side cut graph result shows that nHA@SF is loose, 5SF/5P is compact, and the bonding compactness between the two layers is high.

The nanofiber membrane thickness was measured and averaged, and the data obtained are shown in table 1 below. The double-layer structure is realized and the thickness is smaller, so that the periodontal tissue can be well attached in the clinical use process.

TABLE 1

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

Characterization of the films produced using infrared (Thermo Fisher, nicoletiN 10), fig. 2 is an infrared spectrum of a nanofiber membrane, wherein (a) inner layer nha@sf nanofiber membrane, (B) outer layer 5SF/5P nanofiber membrane; from the figure, it can be seen that the chemical composition of the silk fibroin-based nanofiber bilayer membrane can be verified based on the position and intensity of the absorption peak. Specifically, all Silk Fibroin (SF) -containing nanofiber membranes were at 1623cm ^-1 、1518cm ^-1 And 1233cm ^-1 The part shows obvious effectCorresponds to the absorption peaks of Silk-I and Silk-II (N-H) and Silk-III (C-N) in SF, respectively. After loading nano-hydroxyapatite (nHA), the nHA/SF and PLCL/SF/nHA composite membrane was at 1045cm ^-1 An absorption peak was present at 601cm indicating the presence of P-O bonds in the membrane ^-1 The absorption peaks appearing at this point are derived from the O-P-O phosphate groups in nHA, and these results confirm that nHA was successfully loaded into the SF/PLCL/nHA composite membrane. Furthermore, at 1752cm ^-1 Strong absorption peaks were observed, corresponding to typical asymmetric stretching of polylactic acid (PLA) ester carbonyl groups. At 2995cm ^-1 And 2945cm ^-1 The absorption peaks of (a) respectively belong to asymmetric and symmetric C-H stretching vibration of PLA. 1454cm ^-1 And 1382cm ^-1 The absorption peaks of (2) belong to the C-H deformations of PLA both symmetrically and asymmetrically curved. 1181cm ^-1 、1130cm ^-1 And 1085cm ^-1 The peak at this point is due to the C-O stretching of PLA. From the above, it can be concluded from FTIR spectrum analysis: the prepared silk fibroin-based nanofiber double-layer membrane has basic characteristic groups of constituent substances, and nHA is successfully loaded.

The films produced were characterized by X-ray diffraction (Rigaku, D/max-2550 PC) and the results are shown in fig. 3, where it can be seen that two main characteristic diffraction peaks appear in the XRD pattern of the pure PLCL material, located at 2θ=16.7° and 19.0 °, respectively, indicating the presence of a typical semi-crystalline structure in the PLCL. This is because the solvent used for PLCL is HFIP, which is a benign solvent for PLCL. In dilute solution, the PLCL molecular chains can be completely surrounded by HFIP molecules, the molecular chains are irrelevant to each other, the high polymer exists in the form of isolated coils in the solution, the contact between polymers is reduced, the molecular chains are completely stretched, mutual entanglement is avoided, and the stretched polymer chains tend to be orderly arranged. Whereas fibrous films doped with SF and nHA did not spike, indicating that these composites did not have a distinct crystal structure. The PLCL and SF are mostly present in the fibrous membrane as amorphous structures, with only a small portion of the ordered structures exhibiting a broad peak form. After PLCL and SF are compounded, the ordered structure of part of the polymer chains is disturbed.

The contact angle of the prepared film was measured by a contact angle measuring instrument (Kruss, DSA-30), and as shown in FIG. 4, it can be seen that the contact angles of 10SF, 8SF/2P, 5SF/5P and nHA@SF were 74.76++3.22°, 98.76++1.72°, 108.69 ++2.49°, 67.72 ++2.29°, respectively. From the experimental results, it can be obtained that the hydrophobicity of the silk fibroin-based nanofiber double-layer membrane increases as the SF content ratio decreases. Because the hydroxyl in SF can form hydrogen bond with water molecules, the surface of the membrane has more obvious hydrophilicity. Meanwhile, nHA@SF is most hydrophilic because the surface roughness is large due to the addition of nHA, and the layer film has larger porosity per se due to the parameter setting when the nHA@SF is prepared by electrospinning. The film material has different roughness and surface area, and the resulting surface tension will be different, which has an effect on the contact angle. Generally, when the contact angle is less than 90 °, the larger active area and surface tension of the roughened surface are greater and wetting is easier. Experimental results show that the contact angles of 10SF and nHA@SF are smaller than 90 degrees, and the contact angles of 8SF/2P and 5SF/5P are close to 90 degrees, so that the inner layer and the outer layer of the double-layer membrane prepared by the experiment have good hydrophilicity, and the double-layer membrane has a certain degree of assistance to the adhesion, growth and propagation of cells on the surface of the membrane.

The silk fibroin-based three-dimensional structure bilayer films obtained in examples 1-3 were tested for mechanical properties using a computer tensile and compressive tester (Shanghai Hengzhi, HY-940 FS), as shown in FIG. 5. Wherein (A) stress-strain curve, (B) breaking strength, (C) elongation at break, (D) Young's modulus; the addition of a suitable amount of PLCL can significantly enhance the fracture toughness of the fibrous membrane. The maximum tensile strength of the group of nHA@SF-10SF containing no PLCL is 2.576MPa, while the ratio of SF to PLCL is 5:5 nHA@SF-5SF/5P, the maximum tensile strength being 1.455MPa, and the PLCL weakens the tensile strength to some extent. According to the experimental result, the mechanical property of the silk fibroin nanofiber composite membrane can be improved by adding a proper amount of PLCL, and the mechanical property of the silk fibroin nanofiber composite membrane can be negatively influenced by excessive PLCL. Therefore, for the preparation of silk fibroin nanofiber composite membranes, it is necessary to properly control the addition amount of PLCL while ensuring mechanical properties.

Example 4

(1) The preparation method of the porous loose membrane comprises the following steps: the lyophilized silk fibroin and nano hydroxyapatite particles were dissolved in 10mL Hexafluoroisopropanol (HFIP) at a mass ratio of 4:1 to prepare a mixed solution with a mass-volume ratio of 15%. Dispersing by ultrasonic oscillation for 30min, and stirring for 24 hours by a magnetic stirrer at room temperature to obtain milky SF/nHA electrostatic spinning solution. The SF/nHA electrostatic spinning solution is transferred to a 10mL medical injector in batches, a needle head with the model of 20G is connected to the injector, then the injector is placed in a propulsion pump, a high-voltage generator is connected, the voltage is set to be 15kV, the speed of the propulsion pump is set to be 1.0mL/h, an electrostatic spinning roller is placed below the propulsion pump to receive fibers, the rotating speed is 1000r/min, and the receiving distance is fixed to be 12cm. The whole process is carried out at room temperature with the air humidity of 15-20%, and the prepared monolayer film is named nHA@SF.

(3) The preparation method of the silk fibroin-based nanofiber double-layer membrane comprises the following steps: spinning the SF/PLCL layer compact film on the basis of the porous loose film formed by the SF/nHA layer to form the silk fibroin-based three-dimensional structure double-layer film. The prepared double-layer films are named nHA@SF-8SF/2P respectively. And after spinning, placing the electrospun membrane in a vacuum drying oven for 48 hours to remove the residual organic solvent on the membrane, then crosslinking the membrane in an EDC-NHS system for 4 hours, washing the residual crosslinking agent with deionized water to clean the solvent, and placing the cleaned solvent in a freeze dryer to finally dry the silk fibroin-based three-dimensional structure double-layer membrane.

Example 5

(1) The preparation method of the porous loose membrane comprises the following steps: the lyophilized silk fibroin and nano hydroxyapatite particles were dissolved in 10mL Hexafluoroisopropanol (HFIP) at a mass ratio of 20:3 to prepare a mixed solution with a mass-volume ratio of 15%. Dispersing by ultrasonic oscillation for 30min, and stirring for 24 hours by a magnetic stirrer at room temperature to obtain milky SF/nHA electrostatic spinning solution. The SF/nHA electrostatic spinning solution is transferred to a 10mL medical injector in batches, a needle head with the model of 20G is connected to the injector, then the injector is placed in a propulsion pump, a high-voltage generator is connected, the voltage is set to be 15kV, the speed of the propulsion pump is set to be 1.0mL/h, an electrostatic spinning roller is placed below the propulsion pump to receive fibers, the rotating speed is 1000r/min, and the receiving distance is fixed to be 12cm. The whole process is carried out at room temperature with the air humidity of 15-20%, and the prepared monolayer film is named nHA@SF.

Scanning electron microscope (Hitachi, TM-1000) images of the inner nanofiber membranes prepared in example 3, example 4 and example 5 are shown in A, B, C of FIG. 6, respectively, it is observed that nHA is doped between the fibers, and the nHA content in example 4 is the greatest, but the fibers are thinner compared with example 3, and it is presumed that the nHA content is too much to block the electrostatic spinning needle, so that the fiber diameter is smaller; whereas example 5 has too low an nHA content compared to example 3, so that nHA is unevenly distributed in the fibers. In summary, the ratio of SF to nHA is most suitable in example 3.

Comparative example 1

The only difference from example 1 is that step (3) is not included in the preparation process, i.e. no crosslinking occurs.

Comparative example 2

The difference from example 1 is only that hexafluoroisopropanol, which is the solvent used in step (1) and step (2), is replaced with trifluoroacetic acid.

Comparative example 3

The difference from example 1 is only that the crosslinker EDC-NHS system in step (3) is replaced by tannic acid.

The films obtained in example 2 and comparative example 1 were subjected to in vitro degradation tests using artificial saliva (ph=6.8) to simulate the degradation process of silk fibroin-based nanofiber bilayer films. The crosslinked nHA@SF-8SF/2P and uncrosslinked nHA@SF-8SF/2P samples were cut to a size of 2.5 cm. Times.2.5 cm and the mass thereof was recorded as W after drying and weighing ₀ . Subsequently, the samples were transferred to 5mL of artificial saliva and placed on a shaking table at 37 ℃. At 3, 7, 14, 21, 28 and 49d, after taking out the sample, it was rewashed several times with deionized water and lyophilized, at which point the mass was noted as W. Calculating the residual mass of the sample by a formulaPercentage (Remaining Mass): remaining Mass (%) = (W/W) ₀ )×100％。

The test results are shown in fig. 7. It can be seen that the crosslinked nHA@SF-8SF/2P bilayer membrane has a relatively smooth degradation curve as a whole, and the degradation of the bilayer membrane is maintained at more than 75% after 7 weeks of degradation. While the uncrosslinked nHA@SF-8SF/2P bilayer film was substantially completely degraded in the first week. The use of the cross-linking agent has a great effect on the degradation performance of the material.

SEM observation was performed on the films obtained in example 2 and comparative example 2, and the test method was: 8SF/2P sample of hexafluoroisopropanol as the electrospinning solvent and 8SF/2P sample of trifluoroacetic acid as the solvent were cut into 1.0cm by 1.0cm square shapes. And then selecting a proper scanning electron microscope sample stage, fixing the cut sample on the sample stage by using conductive adhesive, and spraying gold on the sample for 60 seconds in a direct current 4mA state under a certain vacuum degree, so that the surface of the sample has conductivity. Finally, placing the camera under a scanning electron microscope for shooting.

As shown in fig. 8, (a) the solvent was hexafluoroisopropanol and (B) the solvent was trifluoroacetic acid, it can be seen that a relatively continuous, uniform-size fiber structure was formed when the electrospinning solvent was hexafluoroisopropanol, a complete fiber structure was not formed when the electrospinning solvent was trifluoroacetic acid, and droplets were dropped.

The films obtained in example 1 and comparative example 3 were observed.

The test results are shown in fig. 9, wherein (a) the cross-linking agent is tannic acid and (B) the cross-linking agent is EDC-NHS, and it can be seen that the film with tannic acid as the cross-linking agent has rough surface after dynamic drying and is easy to break after contact; the cross-linking agent is EDC-NHS, and has a smoother surface and is difficult to break after being folded and bent for many times.

The applicant states that the present invention is illustrated by the above examples as a silk fibroin-based three-dimensional structure bilayer membrane for periodontal regeneration, and a method for preparing the same and application thereof, but the present invention is not limited to the above examples, i.e., it does not mean that the present invention must be practiced depending on the above examples. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of selected raw materials, addition of auxiliary components, selection of specific modes, etc. fall within the scope of the present invention and the scope of disclosure.

Claims

1. The silk fibroin-based three-dimensional structure double-layer membrane for periodontal regeneration is characterized by comprising a porous loose layer and an outer compact layer, wherein the porous loose layer is a nanofiber membrane composed of silk fibroin and nano hydroxyapatite, the compact 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 compact layer.

2. The silk fibroin-based three-dimensional structure bilayer membrane for periodontal regeneration according to claim 1, wherein the mass ratio of nano hydroxyapatite in the porous loose layer is 10% -30%;

preferably, the particle size of the nano hydroxyapatite is 20-40 nm.

3. The silk fibroin-based three-dimensional structured bilayer membrane for periodontal regeneration according to claim 1 or 2, wherein the weight percentage of silk fibroin in the dense layer is 50% to 100%;

4. A silk fibroin-based three-dimensional structure bilayer membrane for periodontal regeneration according to any one of claims 1 to 3, wherein the crosslinking agent used for crosslinking is carbodiimide and hydroxysuccinimide;

preferably, the mass ratio of the carbodiimide salt to the hydroxysuccinimide is 20-30%.

5. The method for producing a silk fibroin-based three-dimensional structure bilayer membrane for periodontal regeneration according to any one of claims 1 to 4, comprising the steps of:

6. The preparation method according to claim 5, wherein the total mass percentage of the silk fibroin and the nano-hydroxyapatite in the solution containing the silk fibroin and the nano-hydroxyapatite in the step (1) is 15% -25%;

preferably, the solvent of the solution containing silk fibroin and nano hydroxyapatite in the step (1) is hexafluoroisopropanol;

preferably, the preparation process of the solution containing the silk fibroin and the nano hydroxyapatite in the step (1) is as follows: adding the freeze-dried silk fibroin and nano hydroxyapatite particles into hexafluoroisopropanol to obtain a mixed solution, carrying out ultrasonic vibration treatment on the mixed solution, and then stirring to obtain a solution containing the silk fibroin and the nano hydroxyapatite;

preferably, the ultrasonic frequency during the ultrasonic oscillation treatment is 19-23KHz, and the treatment time is 10-40min;

preferably, the stirring time is 3-48 hours;

preferably, the voltage is set to be 10-20 kV during the electrostatic spinning in the step (1), the sample injection rate is set to be 0.8-1.5 mL/h, the fiber is received by using a roller, the rotating speed of the roller is 800-3000 r/min, and the receiving distance is 10-15 cm;

the electrostatic spinning in the step (1) is carried out at room temperature, and the air humidity is 15-20%.

7. The preparation method according to claim 5 or 6, wherein the mass percentage of silk fibroin and biodegradable material in the solution containing silk fibroin and biodegradable material in the step (2) is 10% -20%;

preferably, 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;

preferably, the preparation process of the solution containing the silk fibroin and the biodegradable material in the step (2) is as follows: adding silk fibroin and a biodegradable material into hexafluoroisopropanol to obtain a mixed solution, and stirring to obtain a solution containing the silk fibroin and the biodegradable material;

preferably, the stirring time is 3-24 hours;

preferably, the voltage is set to be 10-20 kV during the electrostatic spinning in the step (2), the sample injection rate is set to be 0.8-1.5 mL/h, the fiber is received by using a roller, the rotating speed of the roller is 800-3000 r/min, and the receiving distance is 10-15 cm;

preferably, the electrospinning in step (2) is performed at room temperature with an air humidity of 15-20%.

8. The method of any one of claims 5-7, wherein the cross-linking agent of step (3) is a carbodiimide salt and hydroxysuccinimide;

9. The method of any one of claims 5-8, wherein the crosslinking of step (3) is performed at room temperature for a period of 1-6 hours;

preferably, the crosslinking of step (3) is performed in an organic solvent, which is absolute ethanol;

10. Use of a silk fibroin-based three-dimensional structure bilayer film for periodontal regeneration according to any one of claims 1-4 in the preparation of an oral implant material.