CN112553785B - Double-layer guided tissue regeneration membrane and preparation method thereof - Google Patents

Abstract

The invention relates to a double-layer guided tissue regeneration membrane and a preparation method thereof. The double-layer guided tissue regeneration membrane is prepared by crosslinking a compact layer and a loose layer which are compounded together; the compact layer is prepared by blending polycaprolactone and chitosan and then carrying out electrostatic spinning; the loose layer is prepared by freeze drying type I collagen. The method comprises the following steps: preparing collagen gel liquid from the type I collagen, and freezing and drying to obtain a loose layer; uniformly mixing a polycaprolactone solution and a chitosan solution to obtain a PCL/CS electro-spinning solution, and preparing a compact layer on the surface of the loose layer by using the PCL/CS electro-spinning solution as a spinning solution through an electrostatic spinning method to obtain a double-layer composite membrane; and (4) crosslinking the double-layer composite membrane to prepare the double-layer guided tissue regeneration membrane. The material prepared by the invention has firmer combination between double layers and obviously improved mechanical property; the compact layer is nano-scale superfine fiber, simulates the fiber structure of extracellular matrix, is beneficial to cell growth and can effectively play a barrier role.

Description

Double-layer guided tissue regeneration membrane and preparation method thereof

Technical Field

The invention belongs to the technical field of medical instrument materials, and particularly relates to a double-layer guided tissue regeneration membrane and a preparation method thereof.

Background

In oral surgical procedures, the use of barrier membranes has become a routine method of Guided Bone Regeneration (GBR), Guided Tissue Regeneration (GTR) for the treatment of periodontal and peri-implant bone defects, and for bone augmentation procedures prior to or contemporaneous with implant implantation. The material of the guided tissue regeneration membrane is the core of the repair management, which serves as a physical barrier to form and maintain a regeneration space above the defective tissue, promoting cell migration and growth to form new tissue, while preventing connective and epithelial cells from growing in.

At present, the tissue regeneration guiding materials widely used in clinic can be divided into two main categories of non-degradable and biodegradable materials. The non-degradable materials mainly include polyacetal, polytetrafluoroethylene, silicone film, etc., and although the materials have better biocompatibility with tissues, the materials also have more defects: 1. the material can not be absorbed by tissues and needs to be taken out by secondary operation, so that the chance of wound is increased; 2. non-absorbable membranes present a significant risk of premature exposure, possibly leading to wound infection and secondary consequences of poor bone regeneration; 3. the non-absorbable membrane must be fixed by screws during operation due to its rigidity, hydrophobicity, etc. Degradable materials, such as polylactic acid (PLA), polyglutamic acid (PGA), and other organic polymer materials, have too fast mechanical properties due to accumulation of local acidic degradation products after being implanted into an organism, lack of biological activity, and cannot induce new bone regeneration, resulting in bone defects remaining after the material is degraded, thereby limiting clinical applications thereof. Although natural biological membrane materials such as collagen, gelatin, chitosan and the like have excellent biocompatibility, the natural biological membrane materials have high degradation speed and poor mechanical properties, and easily cause the collapse of the membrane, thereby influencing the regeneration of tissues.

In recent years, electrospinning technology has been widely used to produce fibrous scaffolds that mimic the structure of extracellular matrix (ECM). The nano fiber scaffold prepared by electrostatic spinning can reach micron-scale or even nano-scale, can selectively allow cells to pass through by controlling the diameter, the aperture and the like of the fiber, and is an ideal material for tissue engineering scaffolds. By utilizing the respective advantages of natural polymers and organic polymers, the composite material prepared by electrostatic spinning is widely applied to the aspects of blood vessel construction, skin repair and bone regeneration. Chinese patent application CN104474589A blends and spins raw materials with different in-vivo degradation times through an electrostatic spinning technology to obtain a double-layer film with a compact layer and a potential loose layer; the loose layer of this patent application comprises the polymer that two kinds of degradation rates are different, and the control of loose layer degradation rate needs through the content of two kinds of polymers of regulation and control, and the loose layer in this patent application has following problem: (1) the content of the two macromolecules can not be simply controlled through the spinning speed or the concentration of the spinning solution, and the spinning speed and the concentration of the spinning solution can directly influence the diameter of the fiber, namely the pore diameter and the porosity of the loose layer can be influenced, which is not beneficial to obtaining the loose layer with stable performance; (2) the fibers of the two polymers are not uniformly distributed on the receiver, and the loose layer may generate cavities in the degradation process, which is not favorable for the adhesion of cells, otherwise, the loose layer is also degraded too slowly, which is not favorable for the creeping of cells. Chinese patent application CN110420359A discloses a guided tissue regeneration membrane, comprising a compact layer facing the periosteum and a loose layer facing the bone defect area; the compact layer is made of type I collagen and has a smooth surface; the loose layer is formed by compounding type I collagen and mineralized collagen nano particles, and the interior of the loose layer is of a porous structure; however, the flexibility and the application properties of the dense layer are to be further improved. At present, GTR films put higher requirements on some physical and chemical properties in practical use: such as hydration time, flexibility, application, suture tension resistance, etc., and secondly, GTR films also place higher demands on barrier and tissue regeneration-guiding biological properties.

Polycaprolactone (PCL) is approved by the FDA in the United states for clinical tests by the unique properties of good mechanical property, thermal stability, biodegradability, low toxicity, biocompatibility, good permeability to drugs and the like, and is an important degradable synthetic polymer in the field of biomedical materials at present. The chitosan has good biocompatibility and biodegradability, certain antibacterial property, hydrophilicity and obvious protein affinity, and the degradation product of the chitosan generally has no toxic or side effect on human bodies, does not accumulate in the bodies and has no immunogenicity, so the chitosan has very wide prospects in the field of biomedicine. Examples of applications that have been developed and potentially include only human skin (wound dressings), surgical sutures and bone repair materials, anticoagulants and artificial dialysis membranes, pharmaceutical preparations and drug delivery agents, and the like. The collagen has good biocompatibility, low immunogenicity, biodegradability, strong hydrophilicity, cell adaptability and cell proliferation effect, and various materials for guiding tissue regeneration have been developed by taking the collagen as a main raw material, so that the collagen has important application value in the aspects of repairing muscle bonds, ligaments, periodontal tissues, peritoneum and the like.

Based on the above, the invention combines the advantages and disadvantages of natural biological materials and degradable polymers to prepare the guided tissue regeneration membrane with a double-layer structure by an electrostatic spinning method.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention provides a double-layer guided tissue regeneration membrane and a preparation method thereof. The method of the invention produces the guided tissue regeneration membrane (GTR membrane) which not only can play a role of an isolation barrier, but also has certain guided tissue regeneration performance and good mechanical performance and operability.

The invention provides a double-layer guided tissue regeneration membrane in a first aspect, which is prepared by crosslinking a compact layer and a loose layer which are compounded together; the compact layer is prepared by blending polycaprolactone and chitosan and then carrying out an electrostatic spinning method; the loose layer is prepared by I type collagen through freeze drying.

Preferably, the loose layer is of a porous structure, the average pore diameter of the loose layer is 20-100 mu m, the porosity is 30-50%, and/or the thickness of the loose layer is 0.05-0.35 mm; and/or the compact layer is of an electro-spun ultrafine fiber structure, the average diameter of fibers contained in the compact layer is 100-500 nm, the size of pores formed among the fibers is 1-10 mu m, and/or the thickness of the compact layer is 0.15-0.35 mm.

Preferably, in the dense layer, the mass ratio of the polycaprolactone to the chitosan is (4-8): 1.

The invention provides a preparation method of a double-layer guided tissue regeneration membrane in a second aspect, which comprises the following steps:

s1, preparation of a loose layer: preparing collagen gel liquid from the type I collagen, and then freezing and drying to prepare a loose layer;

s2, preparing a compact layer: uniformly mixing a polycaprolactone solution and a chitosan solution to obtain a PCL/CS electro-spinning solution, and preparing a compact layer on the surface of the loose layer prepared in the step S1 by using the PCL/CS electro-spinning solution as a spinning solution through an electrostatic spinning method, so as to obtain a double-layer composite membrane consisting of the loose layer and the compact layer;

and S3, crosslinking the double-layer composite membrane obtained in the step S2 to obtain the double-layer guided tissue regeneration membrane.

Preferably, the step S1 includes the following sub-steps:

s11, dissolving the type I collagen in purified water to prepare collagen gel liquid; preferably, the mass concentration of the I type collagen in the collagen gel liquid is 0.05-0.25%;

s12, freeze-drying the collagen gel liquid to obtain a loose layer; the freeze drying comprises a pre-freezing stage, a first sublimation stage, a second sublimation stage and a temperature reduction stage, and the process conditions of each stage are as follows:

a pre-freezing stage: the target temperature is-12 to-8 ℃, the speed is 3 to 4.0 ℃/min, and the constant temperature duration is 280 to 320 min;

a first sublimation stage: vacuumizing, wherein the air entrainment is 90-110 Pa, the target temperature is-4 to-2 ℃, the speed is 0.6-0.8 ℃/min, and the constant temperature duration is 1300-1340 min;

the second sublimation stage, the evacuation, 90~110Pa aerify, including five intensification ladders, do respectively:

the temperature is between-1 and 1 ℃, the speed is 0.2 to 0.3 ℃/min, and the constant temperature duration is 110 to 130 min;

the temperature is 8-12 ℃, the speed is 1.0-1.2 ℃/min, and the constant temperature duration is 110-130 min;

the temperature is 18-22 ℃, the speed is 1.0-1.2 ℃/min, and the constant temperature duration is 110-130 min;

the temperature is 28-32 ℃, the speed is 1.0-1.2 ℃/min, and the constant temperature duration is 110-130 min;

38-42 ℃, the speed is 1.0-1.2 ℃/min, the constant temperature duration is as follows: judging the end point every 10 minutes until the end point is qualified; the end point is judged to be less than or equal to 0.9Pa/10 min;

and (3) cooling: cooling to room temperature at a rate of 1.4-1.6 ℃/min;

s13, trimming the loose layer and fixing it on a cylindrical rotary receiver covered with aluminum foil.

Preferably, the step S2 includes the following sub-steps:

s21, adding polycaprolactone into trifluoroethanol and/or hexafluoroisopropanol, stirring and dissolving to obtain polycaprolactone solution; preferably, the concentration of the polycaprolactone solution is 8-25 w/v%;

s22, adding chitosan into trifluoroacetic acid and/or acetic acid, stirring and dissolving to obtain a chitosan solution; preferably, the concentration of the chitosan solution is 5-10 w/v%;

s23, uniformly mixing the polycaprolactone solution and the chitosan solution according to the volume ratio (2-5) to 1 to obtain a PCL/CS electrospinning solution;

s24, coating a cylindrical rotary receiver attached with an aluminum foil in a spinning chamber by using a loose layer, filling a PCL/CS electrospinning solution into a 10mL syringe, and preparing a compact layer on the surface of the loose layer prepared in the step S1 by using the PCL/CS electrospinning solution as the spinning solution through an electrostatic spinning method, thereby obtaining a double-layer composite membrane consisting of the loose layer and the compact layer;

and S25, removing the double-layer composite film and trimming.

Preferably, the humidity in the spinning chamber is 40-50% RH, and the temperature in the spinning chamber is 25 ℃; the inner diameter of the injector is 14.90mm, and the flow rate is 2 mL/h; during spinning, the receiving distance is 15-20 cm, and the voltage of a high-voltage direct-current power supply is 15-25 kV; and/or in the compact layer, the mass ratio of the polycaprolactone to the chitosan is (4-8): 1.

Preferably, the step S3 includes the following sub-steps:

s31, preparing a crosslinking solution with the concentration of 2-30 mmol/L, wherein the crosslinking agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, preferably, the solvent is an ethanol water solution, and more preferably, the mass concentration of the ethanol water solution is 40-80%;

s32, placing the double-layer composite membrane in a cross-linking solution to be cross-linked for 10-60 min at the temperature of 4 ℃;

s33, cleaning the double-layer composite membrane crosslinked in the step S32, and then soaking in an ethanol water solution;

and S34, carrying out vacuum drying on the double-layer composite membrane processed in the step S33 to obtain the double-layer guided tissue regeneration membrane.

Preferably, in step S33: when cleaning, washing with flowing purified water for 10-30 s, repeating for 3-5 times, and then washing with 40-80% ethanol water solution for 3-5 times, wherein each time of washing is 30-60 s; and/or when soaking, the mass concentration of the ethanol water solution is 40-80%, and the soaking time is 12-48 h.

The present invention provides, in a third aspect, a double-layer guided tissue regeneration membrane produced by the production method according to the first aspect of the invention; preferably, the bilayer guided tissue regeneration membrane has one or more of the following properties: the loose layer is of a porous structure, the average pore diameter of the loose layer is 20-100 mu m, and the porosity is 30-50%; the thickness of the loose layer is 0.05-0.35 mm; the compact layer is of an electro-spun superfine fiber structure, the average diameter of fibers contained in the compact layer is 100-500 nm, and the size of pores formed among the fibers is 1-10 mu m; the thickness of the compact layer is 0.15-0.35 mm.

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

(1) the guided tissue regeneration membrane prepared by an electrostatic spinning method is constructed, one side is a compact layer, and the other side is a loose layer, wherein the compact layer is nano-scale superfine fiber, the fiber structure of extracellular matrix is simulated, the growth of cells is facilitated, and the migration of the cells to the interior of the material is hindered by the undersized aperture and the compact fiber structure, so that the barrier effect is achieved; the loose layer faces to the bone defect area, has certain pore diameter and porosity, has good osteoconductivity, is beneficial to the adhesion and growth of cells, promotes the growth of new bones into the material, and is beneficial to the transportation of nutrient components and the discharge of metabolites.

(2) The compact layer of the guided tissue regeneration membrane prepared by the electrostatic spinning method is composed of PCL and chitosan, the PCL has good mechanical property, thermal stability, biodegradability, low toxicity and biocompatibility, the chitosan has good biocompatibility and biodegradability, certain antibacterial property, hydrophilicity and obvious protein affinity, and degradation products of the chitosan have no toxic or side effect on a human body, do not accumulate in the body and have no immunogenicity; the loose layer is composed of type I collagen, has good biocompatibility, low immunogenicity, biodegradability, strong hydrophilicity, cell adaptability and cell proliferation effect, and can be attached to a diseased oral bone defect surface to promote new bone generation.

(3) In the cross-linking process, amino on the chitosan of the compact layer is combined with carboxyl on the collagen of the loose layer, so that the double layers are combined more firmly; after the guided tissue regeneration membrane prepared by the electrostatic spinning method is crosslinked, the mechanical property of the guided tissue regeneration membrane is obviously improved, and particularly, the tensile strength and the suture tension strength are enhanced.

Drawings

FIG. 1 is a schematic view of an electrospinning apparatus used in the present invention.

FIG. 2 is a scanning electron microscope image of a loose layer included in the double-layer guided tissue regeneration film prepared in example 1 of the present invention. FIG. (a) shows magnification times 80, and FIG. (b) shows magnification times 500.

FIG. 3 is a scanning electron microscope image of a dense layer included in the double-layer guided tissue regeneration membrane prepared in example 1 of the present invention. The magnification is 1000 × for panel (a), and 10000 × for panel (b).

Figure 4 is a photomicrograph of the bulk and dense layers produced in example 1 of the present invention. Fig. (a) shows the loose layer and fig. (b) shows the dense layer.

FIG. 5 is a graph showing the results of the application test of the double-layer guided tissue regeneration membrane prepared in example 1 of the present invention after hydration.

Fig. 6 is a graph of degradation time and mass loss rate of the double-layer guided tissue regeneration membrane prepared in example 1 of the present invention.

FIG. 7 is a schematic view of the present invention in an animal experiment.

FIG. 8 is a graph showing the mucosal growth in the tooth extraction area of group A in the animal experiment of the present invention.

FIG. 9 is a graph showing the observation results of a pathological section of a group A new bone tissue taken 12 weeks after the operation in an animal experiment according to the present invention. The magnification is 200 × for panel (a), and 400 for panel (b).

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

The invention provides a double-layer guided tissue regeneration membrane in a first aspect, which is prepared by crosslinking a compact layer and a loose layer which are compounded together; the compact layer is prepared by blending polycaprolactone and chitosan and then carrying out an electrostatic spinning method; the loose layer is prepared by I type collagen through freeze drying. In the invention, the compact layer is a nano-scale superfine fiber, simulates the fiber structure of extracellular matrix, is beneficial to the growth of cells, and the migration of the cells to the inside of the material is hindered by the undersized aperture and the compact fiber structure, thereby playing a role of barrier; the loose layer faces to the bone defect area, has certain pore diameter and porosity, has good osteoconductivity, is beneficial to the adhesion and growth of cells, promotes the growth of new bones into the material, and is beneficial to the transportation of nutrient components and the discharge of metabolites. In the invention, the loose layer is prepared by freeze drying type I collagen, and is prepared into a loose layer with a pore structure which is beneficial to cell crawling and proliferation by adopting freeze drying, and the degradation of the loose layer is controlled by the crosslinking degree; compared with a potential loose layer which is obtained by CN104474589A through carrying out blended spinning on raw materials with different in-vivo degradation time through an electrostatic spinning technology by using a loose layer prepared by I-type collagen through freeze drying, the degradation rate of the loose layer is easy to control, and the loose layer obtained by the invention has better quality controllability and is more beneficial to cell adhesion and growth. In the invention, the compact layer is prepared by blending polycaprolactone and chitosan and then preparing the mixture by an electrostatic spinning method, and the inventor finds that the single use of chitosan is difficult in electrospinning and has poor mechanical strength; the biocompatibility of polycaprolactone can be improved by adding chitosan.

Chinese patent application CN110420359A discloses a guided tissue regeneration membrane, comprising a compact layer facing the periosteum and a loose layer facing the bone defect area; the compact layer is made of type I collagen and has a smooth surface; the loose layer is formed by compounding type I collagen and mineralized collagen nanoparticles, and the interior of the loose layer is of a porous structure. Compared with the compact layer prepared by a drying mode in CN110420359A, the compact layer prepared by the electrostatic spinning method has better flexibility and application property, and the compact layer can greatly shorten the hydration time and is beneficial to the operation of clinical doctors.

According to some preferred embodiments, the porous layer is a porous structure, the porous layer has an average pore size of 20 to 100 μm (e.g. 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 μm), a porosity of 30 to 50% (e.g. 30%, 35%, 40%, 45% or 50%), and/or a thickness of 0.05 to 0.35mm (e.g. 0.05, 0.1, 0.15, 0.2, 0.25, 0.3 or 0.35 mm); in the present invention, the porous layer has a porous structure, and has good water absorption and good bone conductivity.

According to some preferred embodiments, the dense layer is an electrospun ultrafine fibrous structure, the fibers contained in the dense layer have an average diameter of 100 to 500nm (e.g., 100, 150, 200, 250, 300, 350, 400, 450, or 500nm), a pore size formed between the fibers is 1 to 10 μm, and/or the dense layer has a thickness of 0.15 to 0.35mm (e.g., 0.15, 0.2, 0.25, 0.3, or 0.35 mm). In the invention, the compact layer is a nano-scale superfine fiber, the fiber structure of extracellular matrix is simulated, the growth of cells is facilitated, and the migration of the cells to the interior of the material is hindered by the undersized aperture (1-10 mu m) and the compact fiber structure, so that the effect of an isolation barrier is effectively played.

According to some preferred embodiments, the mass ratio of polycaprolactone to chitosan in the dense layer is (4-8: 1) (e.g., 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, or 8: 1). In the invention, the mass ratio of the polycaprolactone to the chitosan in the compact layer is preferably (4-8): 1, so that the average diameter of fibers contained in the prepared compact layer is effectively ensured to be 100-500 nm, the size of pores formed among the fibers is 1-10 mu m, and the compact layer with the electro-spun superfine fiber structure, which is good in flexibility and good in application property, can be obtained; if the mass ratio of the two is not within this range, the diameter, uniformity and collecting efficiency of the electrospun fibers are adversely affected.

The invention provides a preparation method of a double-layer guided tissue regeneration membrane in a second aspect, which comprises the following steps:

s1, preparation of a loose layer: preparing collagen gel liquid from the type I collagen, and then freezing and drying to prepare a loose layer;

s2, preparing a compact layer: uniformly mixing a polycaprolactone solution and a chitosan solution to obtain a PCL/CS electro-spinning solution, and preparing a compact layer on the surface of the loose layer prepared in the step S1 by using the PCL/CS electro-spinning solution as a spinning solution through an electrostatic spinning method, so as to obtain a double-layer composite membrane consisting of the loose layer and the compact layer;

and S3, crosslinking the double-layer composite membrane obtained in the step S2 to obtain the double-layer guided tissue regeneration membrane.

The invention constructs the guided tissue regeneration membrane with a double-layer structure prepared by an electrostatic spinning method, wherein one side is a compact layer, the other side is a loose layer, the loose layer is prepared by collagen gel through freeze drying, the compact layer is prepared by electrostatic spinning after PCL and chitosan are blended, and after preliminary compounding is completed in the electrostatic spinning process, the composite membrane is crosslinked, so that the firmness degree and the mechanical property of double-layer combination are further improved; in the double-layer guided tissue regeneration membrane, the compact layer is nano-scale superfine fibers, the fibrous structure of extracellular matrix is simulated, the growth of cells is facilitated, and the migration of the cells to the interior of the material is hindered by the undersized pore diameter and the compact fibrous structure, so that a barrier effect is achieved; the loose layer faces the bone defect area, is of a porous structure, has a certain pore diameter and porosity, has good water absorption and good osteoconductivity, is beneficial to cell adhesion and growth, promotes new bones to grow into the material, and is beneficial to transportation of nutrient components and discharge of metabolites; the compact layer of the guided tissue regeneration membrane prepared by the electrostatic spinning method is composed of PCL and chitosan, the PCL has good mechanical property, thermal stability, biodegradability, low toxicity and biocompatibility, the chitosan has good biocompatibility and biodegradability, certain antibacterial property, hydrophilicity and obvious protein affinity, and degradation products of the membrane have no toxic or side effect on a human body, do not accumulate in the body and have no immunogenicity; the compact layer is composed of PCL and chitosan, the PCL and the chitosan have good synergistic effect, the biocompatibility of polycaprolactone can be improved by adding the chitosan, and the defect of poor mechanical strength of the chitosan can be effectively overcome by adding the PCL; the loose layer is composed of type I collagen, has good biocompatibility, low immunogenicity, biodegradability, strong hydrophilicity, cell adaptability and cell proliferation effect, and can be attached to the defected surface of the diseased oral bone to promote the generation of new bone; in the cross-linking process, amino on the chitosan of the compact layer is combined with carboxyl on the collagen of the loose layer, so that the double layers are combined more firmly; after the guided tissue regeneration membrane prepared by the electrostatic spinning method is crosslinked, the mechanical property of the guided tissue regeneration membrane is obviously improved, and particularly, the tensile strength and the suture tension strength are enhanced.

According to some preferred embodiments, said step S1 comprises the following sub-steps:

s11, dissolving the type I collagen in purified water to prepare collagen gel liquid; preferably, the collagen gel solution contains collagen type i at a mass concentration of 0.05% to 0.25% (e.g., 0.05%, 0.08%, 0.1%, 0.15%, 0.2%, or 0.25%);

s12, freeze-drying the collagen gel liquid to obtain a loose layer; the freeze drying comprises a pre-freezing stage, a first sublimation stage, a second sublimation stage and a temperature reduction stage, and the process conditions of each stage are as follows:

a pre-freezing stage: the target temperature is-12 to-8 ℃, preferably-10 ℃, the speed is 3 to 4.0 ℃/min, and the constant temperature duration is 280 to 320min, preferably 300 min;

a first sublimation stage: vacuumizing, wherein the air is mixed at 90-110 Pa, the target temperature is-4 to-2 ℃, preferably-3 ℃, the speed is 0.6-0.8 ℃/min, and the constant temperature duration is 1300-1340 min, preferably 1320 min;

the second sublimation stage, the evacuation, 90~110Pa aerify, including five intensification ladders, do respectively:

the temperature is preferably 0 ℃ below zero at 1-1 ℃, the speed is 0.2-0.3 ℃/min, the constant temperature duration is 110-130 min, and preferably 120 min;

the temperature is preferably 10 ℃ at 8-12 ℃, the speed is 1.0-1.2 ℃/min, the constant temperature duration is 110-130 min, and preferably 120 min;

the temperature is preferably 20 ℃ at 18-22 ℃, the speed is 1.0-1.2 ℃/min, the constant temperature duration is 110-130 min, and preferably 120 min;

the temperature is preferably 30 ℃ at 28-32 ℃, the speed is 1.0-1.2 ℃/min, the constant temperature duration is 110-130 min, and preferably 120 min;

the temperature of 38-42 ℃ is preferably 40 ℃, the speed is 1.0-1.2 ℃/min, the constant temperature duration is as follows: judging the end point every 10 minutes until the end point is qualified; the end point is judged to be less than or equal to 0.9Pa/10 min;

and (3) cooling: cooling to room temperature (for example, room temperature 25 ℃), at a rate of 1.4-1.6 ℃/min;

s13, trimming the loose layer and fixing it on a cylindrical rotary receiver covered with aluminum foil.

According to some preferred embodiments, said step S2 comprises the following sub-steps:

s21, adding polycaprolactone into trifluoroethanol and/or hexafluoroisopropanol, stirring and dissolving to obtain polycaprolactone solution; preferably, the concentration of the polycaprolactone solution is 8-25 w/v% (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 w/v%); in the invention, the concentration of the polycaprolactone solution represents the mass of polycaprolactone contained in the polycaprolactone solution in unit volume, and the unit is w/v%;

s22, adding chitosan into trifluoroacetic acid and/or acetic acid, stirring and dissolving to obtain a chitosan solution; preferably, the concentration of the chitosan solution is 5-10 w/v% (e.g., 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 w/v%); in the present invention, the concentration of the chitosan solution represents the mass of chitosan contained in a unit volume of the chitosan solution, in units of w/v%;

s23, uniformly mixing the polycaprolactone solution and the chitosan solution according to the volume ratio (2-5) to 1, preferably 4:1, to obtain a PCL/CS electrospinning solution;

s24, coating a cylindrical rotary receiver attached with an aluminum foil in a spinning chamber by using a loose layer, filling a PCL/CS electrospinning solution into a 10mL syringe, and preparing a compact layer on the surface of the loose layer prepared in the step S1 by using the PCL/CS electrospinning solution as the spinning solution through an electrostatic spinning method, thereby obtaining a double-layer composite membrane consisting of the loose layer and the compact layer; preferably, the humidity in the spinning chamber is 40-50% RH (relative humidity), and the temperature in the spinning chamber is 25 ℃; the inner diameter of the injector is 14.90mm, and the flow rate is 2 mL/h; during spinning, the receiving distance is 15-20 cm, and the voltage of a high-voltage direct-current power supply is 15-25 kV;

s25, removing the double-layer composite film and trimming; in some embodiments, after the dense layer reaches a certain thickness, the double-layer composite membrane is cut along the seam of the loose layer, for example, by a blade, and taken out; and cutting and trimming the taken out double-layer composite film.

The preparation method preferably adopts the step S2 to include the substeps S21 to S24 to prepare the dense layer, and the experimental parameters influencing the fiber diameter, such as solution concentration, the mass ratio of polycaprolactone to chitosan, the injection rate, the receiving distance, the temperature, the humidity and the like, are optimized, so that the dense layer with the electro-spun ultrafine fiber structure is favorably obtained, the average diameter of fibers contained in the dense layer is 100-500 nm, the size of pores formed among the fibers is 1-10 mu m, the dense layer is made into nanoscale ultrafine fibers, the fiber structure of extracellular matrix is simulated, the cell growth is favorably realized, and the migration of cells to the inside of the material is hindered by the undersized pore diameter (1-10 mu m) and the dense fiber structure, so that the effect of an isolation barrier is effectively achieved.

According to some specific embodiments, the mass ratio of the polycaprolactone to the chitosan in the dense layer is (4-8: 1) (e.g., 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, or 8: 1); in the invention, the mass ratio of polycaprolactone contained in the polycaprolactone solution to chitosan contained in the chitosan solution is preferably (4-8): 1, so that the average diameter of fibers contained in the prepared compact layer is effectively ensured to be 100-500 nm, the size of pores formed among the fibers is 1-10 mu m, and the compact layer with the electro-spun superfine fiber structure meeting the requirement is obtained; if the mass ratio of the two is not within this range, the diameter, uniformity and collecting efficiency of the electrospun fibers are adversely affected.

According to some preferred embodiments, said step S3 comprises the following sub-steps:

s31, preparing a crosslinking solution with a concentration of 2-30 mmol/L (such as 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30mmol/L), wherein the crosslinking agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, the solvent is preferably an aqueous ethanol solution, and the solvent is more preferably an aqueous ethanol solution with a mass concentration of 40-80% (such as 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80%);

s32, placing the double-layer composite membrane in a cross-linking solution to perform cross-linking for 10-60 min (for example, 10, 20, 30, 40, 50 or 60min) at 4 ℃; in the invention, the double-layer composite membrane is placed in a crosslinking liquid for crosslinking at 4 ℃ so as to ensure the activity of the double-layer composite membrane;

s33, cleaning the double-layer composite membrane crosslinked in the step S32, and then soaking in an ethanol water solution;

and S34, performing vacuum drying (for example, vacuum drying at 42 ℃) on the double-layer composite membrane processed in the step S33 to obtain the double-layer guided tissue regeneration membrane.

According to some preferred embodiments, in step S33: when cleaning, washing with flowing purified water for 10-30 s, repeating for 3-5 times, and then washing with 40-80% ethanol water solution for 3-5 times, wherein each time of washing is 30-60 s; and/or when soaking, the mass concentration of the ethanol water solution is 40-80%, and the soaking time is 12-48 h.

In some preferred embodiments, the bilayer guided tissue regeneration membrane prepared by the preparation method of the present invention has one or more of the following properties:

the loose layer is of a porous structure, the average pore diameter of the loose layer is 20-100 mu m, and the porosity is 30-50%;

the thickness of the loose layer is 0.05-0.35 mm;

the compact layer is of an electro-spun superfine fiber structure, the average diameter of fibers contained in the compact layer is 100-500 nm, and the size of pores formed among the fibers is 1-10 mu m;

the thickness of the compact layer is 0.15-0.35 mm.

According to some specific embodiments, the double-layer guided tissue regeneration membrane and the preparation method thereof of the present invention include the steps of:

step S1, preparing a loose layer, which specifically comprises the following steps:

s1-1, dissolving the type I collagen in purified water to prepare collagen gel liquid, wherein the mass concentration of the collagen is 0.05-0.25%;

and S1-2, putting the collagen gel liquid obtained in the S1-1 into a freeze dryer for freeze drying, wherein the freeze drying process is as follows:

(1) cooling the partition plate to-10 ℃ in advance;

(2) the end point is judged to be less than or equal to 0.9Pa/10 min; the end point judging method comprises the following steps: performing manual end point judgment every 10min for 3 times continuously; the specific procedures of freeze-drying are shown in table 1 below; wherein the set temperature in table 1 indicates the time required to reach the set temperature from the last temperature.

Table 1: the specific steps of the freeze drying process.

Step S1-3: the loose collagen sponge (sponge-like loose layer) prepared in S1-2 was trimmed and fixed on a cylindrical rotating receiver covered with aluminum foil.

S2: the preparation of the compact layer specifically comprises the following steps:

s2-1: preparing a spinning solution:

a. poly (epsilon-caprolactone) (PCL) (Mw 80,000) with certain mass is weighed and added into trifluoroethanol or hexafluoroisopropanol to be stirred and dissolved overnight, thus obtaining poly (epsilon-caprolactone) solution with 8-25 w/v percent.

b. A certain mass of Chitosan (CS) (Mw 200,000) was weighed, added to trifluoroacetic acid or acetic acid, and dissolved overnight with stirring to prepare a 5-10 w/v% Chitosan (CS) solution.

And (3) uniformly stirring and mixing 4mL of poly (epsilon-caprolactone) solution and 1mL of chitosan solution to prepare PCL/CS electrospinning solution for later use.

S2-2: preparation of PCL/CS electrospun fiber membrane

The humidity of the spinning chamber is controlled to be maintained at 40-50% by adjusting a humidifier and a dehumidifier of the spinning chamber, and the temperature in the spinning chamber is maintained at about 25 ℃ by adjusting an air conditioner. The micro syringe pump was placed on the side of the cylindrical rotating receiver while the receiver and high voltage power supply were connected to ground. The PCL/CS electrostatic spinning solution is filled into a 10mL injector, the injector is fixed on a micro injection pump and is provided with parameters, the inner diameter of the injector is 14.90mm, the flow rate is set to be 2mL/h, and a chuck connected with a high-voltage direct-current power supply is connected with a needle head of the injector. And covering the cylindrical rotating receiver attached with the aluminum foil by using a loose layer, adjusting the needle head of the injector to be aligned to the center of the cylindrical receiver, wherein the receiving distance is 15-20 cm, and adjusting the voltage of a high-voltage direct-current power supply to be 15-25 kV.

S2-3: taking down the double-layer composite film

After the compact layer reaches a certain thickness, the double-layer composite membrane is scratched by a blade along the seam of the loose layer and taken out.

S2-4: pruning

And cutting the double-layer composite film obtained in the S2-3.

S3: crosslinking, washing and drying of double-layer composite membrane

Step S3-1, preparing 2mM-30mM ethanol aqueous solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) (the concentration of the ethanol aqueous solution is 40 wt% -80 wt%) as a cross-linking agent;

s3-2, placing the double-layer composite membrane obtained in the S2-4 in a cross-linking liquid, and preventing cross-linking for 10-60 min in a refrigerator at 4 ℃;

step S3-3, taking out the double-layer composite membrane obtained in the step S3-2 from the cross-linking agent, washing the double-layer composite membrane for 10S with flowing purified water, repeating the washing for three times, washing the membrane for three times with 40-80 wt% ethanol water solution for 30S each time, and then soaking the membrane in 40-80 wt% ethanol for 12-48h to remove residual cross-linking agent;

and S3-4, carrying out vacuum drying on the double-layer composite membrane obtained in the step S3-3 to obtain the double-layer guide tissue regeneration membrane prepared by the electrostatic spinning method.

The present invention provides, in a third aspect, a double-layer guided tissue regeneration membrane produced by the production method according to the first aspect of the present invention.

The invention will be further illustrated by way of example, but the scope of protection is not limited to these examples.

Example 1

Step S1, preparing a loose layer, which specifically comprises the following steps:

s1-1, dissolving the type I collagen in purified water to prepare collagen gel liquid, wherein the mass concentration of the collagen is 0.1%;

step S1-2, putting the collagen gel liquid obtained in the step S1-1 into a polytetrafluoroethylene plate, uniformly casting, and putting into a freeze dryer for freeze drying, wherein the freeze drying process comprises the following steps:

(1) cooling the partition plate to-10 ℃ in advance;

(2) the end point is judged to be less than or equal to 0.9Pa/10 min; the end point judging method comprises the following steps: performing manual end point judgment every 10min for 3 times continuously; the specific procedures for freeze-drying were performed according to the invention as shown in Table 1.

Step S1-3: the loose collagen sponge (sponge-like loose layer) prepared in S1-2 was trimmed and fixed on a cylindrical rotating receiver covered with aluminum foil.

S2: the preparation of the compact layer specifically comprises the following steps:

s2-1: preparing a spinning solution:

a. a certain mass of poly (e-caprolactone) (PCL) (Mw 80,000) was weighed, and dissolved overnight in trifluoroethanol or hexafluoroisopropanol with stirring to prepare a 15 w/v% poly (e-caprolactone) solution.

b. A certain mass of Chitosan (CS) (Mw 200,000) was weighed, added to trifluoroacetic acid or acetic acid, and dissolved overnight with stirring to prepare an 8 w/v% Chitosan (CS) solution.

Taking 4mL of poly (epsilon-caprolactone) solution and 1mL of chitosan solution, and uniformly stirring and mixing to prepare PCL/CS electrospinning solution for later use; wherein the mass ratio of the poly (epsilon-caprolactone) contained in the poly (epsilon-caprolactone) solution to the chitosan contained in the chitosan solution is 7.5: 1.

S2-2: preparation of PCL/CS electrospun fiber membrane

The relative humidity of the spinning chamber is controlled to be maintained at 45% RH by adjusting a humidifier and a dehumidifier of the spinning chamber, and the temperature in the spinning chamber is maintained at about 25 ℃ by adjusting an air conditioner. The micro syringe pump was placed on the side of the cylindrical rotating receiver while the receiver and high voltage power supply were connected to ground. The PCL/CS electrostatic spinning solution is filled into a 10mL injector, the injector is fixed on a micro injection pump and is provided with parameters, the inner diameter of the injector is 14.90mm, the flow rate is set to be 2mL/h, and a chuck connected with a high-voltage direct-current power supply is connected with a needle head of the injector. And (3) coating the cylindrical rotating receiver attached with the aluminum foil by using a loose layer, adjusting the needle head of the injector to be aligned to the center of the cylindrical receiver, wherein the receiving distance is 18cm, and adjusting the voltage of the high-voltage direct-current power supply to be 20 kV.

S2-3: taking down the double-layer composite film

After the compact layer reaches a certain thickness, the double-layer composite membrane is scratched by a blade along the seam of the loose layer and taken out.

S2-4: pruning

And cutting the double-layer composite film obtained in the S2-3.

S3: crosslinking, washing and drying of double-layer composite membrane

Step S3-1, preparing 15mM ethanol aqueous solution (ethanol aqueous solution concentration is 60 wt%) of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) as a cross-linking agent;

s3-2, placing the double-layer composite membrane obtained in the S2-4 in a cross-linking liquid, and preventing cross-linking for 30min in a refrigerator at 4 ℃;

step S3-3, taking out the double-layer composite membrane obtained in the step S3-2 from the cross-linking agent, washing the double-layer composite membrane for 10S with flowing purified water, repeating the washing for three times, washing the membrane for three times with 60 wt% ethanol water solution for 30S each time, and then soaking the membrane in 60 wt% ethanol for 24h to remove residual cross-linking agent;

and S3-4, carrying out vacuum drying on the double-layer composite membrane obtained in the step S3-3 at 42 ℃ to obtain the double-layer guide tissue regeneration membrane prepared by the electrostatic spinning method.

Scanning electron micrographs of the loose layer and the dense layer included in the double-layer guided tissue regeneration membrane prepared in this example are shown in fig. 2 and fig. 3, respectively. As can be seen from fig. 2, the loose layer is a porous structure, and the dense layer is an electrospun microfiber structure. In addition, the thickness of the loose layer was 0.2mm, the thickness of the dense layer was 0.24mm, and the total thickness of the double layer guided tissue regeneration membrane was 0.44 mm.

Macroscopic photographs of the bulk and dense layers produced in this example are shown in fig. 4. In the embodiment, after the prepared double-layer guided tissue regeneration membrane is soaked in normal saline for 3min, the double-layer guided tissue regeneration membrane has good flexibility and good operation performance; the double-layer guided tissue regeneration membrane hydrated in this example was subjected to a dressing test, and as shown in fig. 5, the double-layer guided tissue regeneration membrane in this example exhibited excellent dressing properties. The guided tissue regeneration membrane is relatively stiff before being hydrated by normal saline, cannot be effectively implanted into an animal body, and is not beneficial to suture; the invention discovers that the hydration time of the double-layer guide tissue regeneration membrane is only about three minutes, and the hydrated double-layer guide tissue regeneration membrane shows excellent flexibility and application property.

The present invention also prepared a guided tissue regeneration membrane according to the same conditions as in example 1 of chinese patent application CN110420359A, and the guided tissue regeneration membrane was soaked in physiological saline for 6min, so that the membrane showed flexibility and application property comparable to those of the double-layer guided tissue regeneration membrane prepared in this example. Compared with the guided tissue regeneration membrane prepared in CN110420359A, the double-layer guided tissue regeneration membrane prepared in the embodiment has better flexibility and application property, can greatly shorten the hydration time, and is beneficial to the operation of clinical doctors.

Example 2

Example 2 is essentially the same as example 1, except that:

in step S2-1, taking 2.4mL of poly (epsilon-caprolactone) solution and 1mL of chitosan solution, stirring and mixing uniformly to prepare PCL/CS electrospinning solution for later use; wherein the mass ratio of the poly (epsilon-caprolactone) contained in the poly (epsilon-caprolactone) solution to the chitosan contained in the chitosan solution is 4.5: 1.

Example 3

Example 3 is essentially the same as example 1, except that:

in step S2-1, 3.2mL of poly (epsilon-caprolactone) solution and 1mL of chitosan solution are taken and stirred and mixed uniformly to prepare PCL/CS electrospinning solution for later use; wherein the mass ratio of the poly (epsilon-caprolactone) contained in the poly (epsilon-caprolactone) solution to the chitosan contained in the chitosan solution is 6: 1.

Comparative example 1

Comparative example 1 is substantially the same as example 1 except that:

in step S2-1, 0.5mL of poly (epsilon-caprolactone) solution and 1mL of chitosan solution are taken and stirred and mixed uniformly to prepare PCL/CS electrospinning solution for later use; wherein the mass ratio of the poly (epsilon-caprolactone) contained in the poly (epsilon-caprolactone) solution to the chitosan contained in the chitosan solution is 0.94: 1.

In step S2-2, the flow rate was 1mL/h, the take-up distance during spinning was 12cm, and the voltage of the high voltage DC power supply was 10 kV.

Comparative example 2

Comparative example 2 is substantially the same as example 1 except that:

in step S2-1, taking 6mL of poly (epsilon-caprolactone) solution and 1mL of chitosan solution, stirring and mixing uniformly to prepare PCL/CS electrospinning solution for later use; wherein the mass ratio of the poly (epsilon-caprolactone) contained in the poly (epsilon-caprolactone) solution to the chitosan contained in the chitosan solution is 11: 1.

In step S2-2, the flow rate was 3mL/h, the take-up distance during spinning was 25cm, and the voltage of the high voltage DC power supply was 30 kV.

Comparative example 3

Comparative example 3 is substantially the same as example 1 except that:

s2: the preparation of the compact layer specifically comprises the following steps:

s2-1: preparing a spinning solution:

a certain mass of poly (e-caprolactone) (PCL) (Mw 80,000) was weighed, and dissolved overnight in trifluoroethanol or hexafluoroisopropanol with stirring to prepare a 15 w/v% poly (e-caprolactone) solution.

5mL of poly (epsilon-caprolactone) solution was taken as PCL electrospinning solution for future use.

S2-2: preparation of electrospun fibrous membranes

The relative humidity of the spinning chamber is controlled to be maintained at 45% RH by adjusting a humidifier and a dehumidifier of the spinning chamber, and the temperature in the spinning chamber is maintained at about 25 ℃ by adjusting an air conditioner. The micro syringe pump was placed on the side of the cylindrical rotating receiver while the receiver and high voltage power supply were connected to ground. And (3) filling the PCL electrospinning solution into a 10mL syringe, fixing the syringe on a micro injection pump, setting parameters, setting the inner diameter of the syringe to be 14.90mm, setting the flow rate to be 2mL/h, and connecting a chuck connected with a high-voltage direct-current power supply with a needle of the syringe. And (3) coating the cylindrical rotating receiver attached with the aluminum foil by using a loose layer, adjusting the needle head of the injector to be aligned to the center of the cylindrical receiver, wherein the receiving distance is 18cm, and adjusting the voltage of the high-voltage direct-current power supply to be 20 kV.

Comparative example 4

Comparative example 4 is substantially the same as example 1 except that:

s2-1: preparing a spinning solution:

b. a certain mass of Chitosan (CS) (Mw 200,000) was weighed, added to trifluoroacetic acid or acetic acid, and dissolved overnight with stirring to prepare an 8 w/v% Chitosan (CS) solution.

5mL of chitosan solution is taken as CS electrospinning solution for later use.

S2-2: preparation of PCL/CS electrospun fiber membrane

The relative humidity of the spinning chamber is controlled to be maintained at 45% RH by adjusting a humidifier and a dehumidifier of the spinning chamber, and the temperature in the spinning chamber is maintained at about 25 ℃ by adjusting an air conditioner. The micro syringe pump was placed on the side of the cylindrical rotating receiver while the receiver and high voltage power supply were connected to ground. And (3) filling the CS electrospinning solution into a 10mL syringe, fixing the syringe on a micro injection pump, setting parameters, setting the inner diameter of the syringe to be 14.90mm, setting the flow rate to be 2mL/h, and connecting a chuck connected with a high-voltage direct-current power supply with a needle of the syringe. And (3) coating the cylindrical rotating receiver attached with the aluminum foil by using a loose layer, adjusting the needle head of the injector to be aligned to the center of the cylindrical receiver, wherein the receiving distance is 18cm, and adjusting the voltage of the high-voltage direct-current power supply to be 20 kV.

The performance of the double-layer guided tissue regeneration membranes prepared in examples 1 to 3 and comparative examples 1 to 4 was tested.

1. And (3) testing tensile strength: basis of tensile Strength test

ASTM-D882-02 (Chinese) Plastic sheet material tensile resistance standard method, sample film is cut into 150mm x 20mm rectangular samples, the samples are put on the tester to carry on the tensile strength test, the experimental conditions are: the anchor clamps interval is 100mm, anchor clamps separating speed: 50mm/min and the initial strain rate is 50 mm/min. The samples in the examples and comparative examples were sterilized with Co60 at a dose of 15KGy, and tensile strength tests were performed after sterilization, respectively, and the experimental conditions are shown in table 2; the results of the experiment are shown in Table 3.

Table 2: tensile strength test conditions.

HThickness of/mm	BWidth of/mm	A0 area/mm2	L0 distance between the clamps/mm	LOriginal length/mm
					0.44	20	1.60	100.00	100.00

Table 3: tensile strengths of examples 1-3 and comparative examples 1-4.

Examples	Tensile Strength/N	Elongation at break/%
			Example 1	12.61±1.21	4.83±0.70
Example 2	5.63±0.98	4.22±0.56
			Example 3	7.64±0.75	4.59±0.81
Comparative example 1	0.93±0.45	4.06±0.69
			Comparative example 2	14.27±1.81	5.92±0.87
Comparative example 3	17.38±2.11	6.53±0.88
			Comparative example 4	0.59±0.35	3.78±0.65

2. Tensile strength test of the anti-suture line: the tensile strength test conditions of the anti-suture line are as follows: anchor clamps interval 100mm, anchor clamps separation speed: 50mm/min, initial strain rate: 0.5 mm/min. A non-absorbable suture of # 3-0 was used, and the sample was threaded through the center with a twin strand 75mm long.

The samples of the examples and comparative examples were sterilized with Co60 at a dose of 15KGy, and the results of the test were measured as shown in table 4, after sterilization, and the suture tension strength test was performed.

Table 4: the suture tensile strengths of examples 1-3 and comparative examples 1-4.

3. And (3) testing the degradation performance: control sample: refer to the material prepared by the preparation method provided in example 1 in application publication No. CN 110420359A; experimental samples: the sample prepared in inventive example 1. 20 films with approximate sizes and thicknesses are respectively selected from the experimental sample and the control sample, the films are respectively weighed and recorded as W0, each film is independently soaked in 2% glycine aqueous solution by mass fraction for 24 hours, then the films are taken out, each film is independently placed into a 15mL centrifuge tube, 10mL of 50Unit/mL collagen hydrolase solution is added, and the reaction degradation is carried out in a constant temperature and humidity box at 37 +/-1 ℃ until the sample structure is completely disintegrated. Taking out two membranes respectively in the reaction periods of 2h, 4h, 8h, 12h, 24h, 36h, 48h, 60h, 72h, 84h and 96h, washing the two membranes with purified water, wiping the surfaces with quantitative filter paper, drying in vacuum to constant weight, and weighing with an analytical balance to obtain W1; if the sample is structurally damaged and does not have a 3D structure in the experimental process, the degradation experiment is immediately stopped, the sample is taken out of the solution containing the collagenase, the quantitative filter paper is filtered and washed, the sample is dried in vacuum to constant weight, and the W2 is obtained by weighing the total mass of the filter paper and the residue through an analytical balance.

The mass loss rate is calculated by the formula (W0-W1)/W0X 100%,

the end point mass loss rate is calculated by the formula (W0-W2)/W0X 100%.

The test results are shown in table 5.

The data in table 5 above were used to generate a degradation time versus mass loss curve, the results of which are shown in fig. 6. As can be seen from table 5 and fig. 6, the degradation rate of the experimental sample is slow in the first 96h, the mass loss rate only reaches 13.67% in 96h, while the control sample starts to degrade slowly and starts to degrade rapidly after 48h, structural damage occurs in 96h, and the mass loss rate reaches 66.89%, and the experimental sample does not have a 3D structure. Therefore, the degradation period of the double-layer guide tissue regeneration membrane prepared by the invention is longer, the degradation of collagen components and chitosan components is mainly performed at the early stage, and the PCL component begins to be gradually degraded along with the extension of the implantation time.

4. Animal experiments:

the third premolar on both sides of the mandible of 4 dogs was removed, and there were 16 sockets in total, which were randomly divided into three groups A, B, and C, each of which was 6, 5, and 5 sockets. Implanting nano hydroxyapatite/collagen material into the alveolus of group A, covering the surface with the GTR film in the embodiment 1 of the invention, implanting artificial bone repair material into the alveolus of group B, covering the surface with a novel composite mineralized collagen film, leaving the alveolus of group C empty (control), and tightly suturing the alveolus; the schematic diagram of the present invention when animal experiments are performed is shown in fig. 7. Preparing experimental samples 12 weeks after the operation, and carrying out gross observation, section staining and the like; the growth condition of the mucous membrane in the tooth extraction area of the group A is shown in fig. 8, and it can be seen from fig. 8 that the mucous membrane in the tooth extraction area of the experimental group A grows well, the alveolar ridge in the tooth extraction area is hard in texture, and no material residue is seen; the pathological section observation results of the group a new bone tissues taken 12 weeks after the operation are shown in fig. 9, and it can be seen from fig. 9 that the bone cells and osteoblasts have high maturity, dense numbers, and a large number of trabeculae have grown into the gaps between the materials.

According to the invention, after the loose layer and the compact layer are compounded, the overall tensile strength of the suture is enhanced, the suture is not easy to tear, and the flexibility and the application performance are obviously improved. The prepared double-layer guide tissue regeneration membrane can be applied to treatment of periodontal defects/bone defects, alveolar ridge increment, guide of bone defect regeneration occurring in immediate planting and delayed planting, treatment of bone absorption and maxillary sinus lifting caused by peri-implantitis, and good overall clinical effect performance.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although described in detail with reference to the foregoing embodiments, those of ordinary skill in the art will understand that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims (9)

1. A preparation method of a double-layer guided tissue regeneration membrane is characterized by comprising the following steps:

s1, preparation of a loose layer: preparing collagen gel liquid from the type I collagen, and then freezing and drying to prepare a loose layer;

s2, preparing a compact layer: uniformly mixing a polycaprolactone solution and a chitosan solution to obtain a PCL/CS electro-spinning solution, and preparing a compact layer on the surface of the loose layer prepared in the step S1 by using the PCL/CS electro-spinning solution as a spinning solution through an electrostatic spinning method, so as to obtain a double-layer composite membrane consisting of the loose layer and the compact layer; in the compact layer, the mass ratio of polycaprolactone to chitosan is (4-8): 1;

s3, crosslinking the double-layer composite membrane obtained in the step S2 to obtain a double-layer guided tissue regeneration membrane;

wherein, step S1 includes the following substeps:

s11, dissolving the type I collagen in purified water to prepare collagen gel liquid;

s12, freeze-drying the collagen gel liquid to obtain a loose layer; the freeze drying comprises a pre-freezing stage, a first sublimation stage, a second sublimation stage and a temperature reduction stage, and the process conditions of each stage are as follows:

a pre-freezing stage: the target temperature is-12 to-8 ℃, the speed is 3 to 4.0 ℃/min, and the constant temperature duration is 280 to 320 min;

a first sublimation stage: vacuumizing, wherein the air is mixed at 90-110 Pa, the target temperature is-4 to-2 ℃, the speed is 0.6-0.8 ℃/min, and the constant temperature duration is 1300-1340 min;

the second sublimation stage, the evacuation, 90~110Pa aerify, including five intensification ladders, do respectively:

the temperature is between-1 and 1 ℃, the speed is 0.2 to 0.3 ℃/min, and the constant temperature duration is 110 to 130 min;

the temperature is 8-12 ℃, the speed is 1.0-1.2 ℃/min, and the constant temperature duration is 110-130 min;

the temperature is 18-22 ℃, the speed is 1.0-1.2 ℃/min, and the constant temperature duration is 110-130 min;

the temperature is 28-32 ℃, the speed is 1.0-1.2 ℃/min, and the constant temperature duration is 110-130 min;

38-42 ℃, the speed is 1.0-1.2 ℃/min, the constant temperature duration is as follows: judging the end point every 10 minutes until the end point is qualified; the end point is judged to be less than or equal to 0.9Pa/10 min;

and (3) cooling: cooling to room temperature at a rate of 1.4-1.6 ℃/min;

s13, trimming the loose layer and fixing the loose layer on a cylindrical rotary receiver coated with aluminum foil;

wherein, step S2 includes the following substeps:

s21, adding polycaprolactone into trifluoroethanol and/or hexafluoroisopropanol, stirring and dissolving to obtain polycaprolactone solution; the concentration of the polycaprolactone solution is 8-25 w/v%;

s22, adding chitosan into trifluoroacetic acid and/or acetic acid, stirring and dissolving to obtain a chitosan solution; the concentration of the chitosan solution is 5-10 w/v%;

s23, uniformly mixing the polycaprolactone solution and the chitosan solution according to the volume ratio (2-5) to 1 to obtain a PCL/CS electrospinning solution;

s24, coating a cylindrical rotary receiver attached with an aluminum foil in a spinning chamber by using a loose layer, filling a PCL/CS electrospinning solution into a 10mL syringe, and preparing a compact layer on the surface of the loose layer prepared in the step S1 by using the PCL/CS electrospinning solution as the spinning solution through an electrostatic spinning method, thereby obtaining a double-layer composite membrane consisting of the loose layer and the compact layer; the inner diameter of the injector is 14.90mm, and the flow rate is 2 mL/h; during spinning, the receiving distance is 15-20 cm, and the voltage of a high-voltage direct-current power supply is 15-25 kV;

and S25, removing the double-layer composite film and trimming.

2. The method of claim 1, wherein:

in step S11, the collagen gel solution contains collagen type i at a mass concentration of 0.05% to 0.25%.

3. The method of claim 1, wherein:

the humidity in the spinning chamber is 40-50% RH, and the temperature in the spinning chamber is 25 ℃.

4. The method for preparing a composite material according to claim 1, wherein the step S3 includes the following sub-steps:

s31, preparing a crosslinking solution with the concentration of 2-30 mmol/L, wherein the crosslinking agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride;

s32, placing the double-layer composite membrane in a cross-linking solution to perform cross-linking for 10-60 min at 4 ℃;

s33, cleaning the double-layer composite membrane crosslinked in the step S32, and then soaking in an ethanol water solution;

and S34, carrying out vacuum drying on the double-layer composite membrane processed in the step S33 to obtain the double-layer guided tissue regeneration membrane.

5. The method of claim 4, wherein:

in the crosslinking solution of step S31, the solvent is an aqueous ethanol solution.

6. The method of claim 5, wherein:

in the crosslinking solution in step S31, the solvent is an ethanol aqueous solution with a mass concentration of 40-80%.

7. The production method according to claim 4, wherein in step S33:

when cleaning, washing with flowing purified water for 10-30 s, repeating for 3-5 times, and then washing with 40-80% ethanol water solution for 3-5 times, wherein each time of washing is 30-60 s; and/or

When the soaking is carried out, the mass concentration of the ethanol water solution is 40-80%, and the soaking time is 12-48 h.

8. The double-layer guided tissue regeneration membrane produced by the production method according to any one of claims 1 to 7.

9. The bilayer guided tissue regeneration membrane of claim 8, wherein the bilayer guided tissue regeneration membrane has one or more of the following properties:

the loose layer is of a porous structure, the average pore diameter of the loose layer is 20-100 mu m, and the porosity is 30-50%;

the thickness of the loose layer is 0.05-0.35 mm;

the compact layer is of an electro-spun ultrafine fiber structure, the average diameter of fibers contained in the compact layer is 100-500 nm, and the size of pores formed among the fibers is 1-10 mu m;

the thickness of the compact layer is 0.15-0.35 mm.

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