A double-layer guided tissue regeneration membrane and preparation method thereof

Abstract

This invention discloses a bilayer guided tissue regeneration membrane and its preparation method. The bilayer guided tissue regeneration membrane comprises a dense layer and a loose layer. The dense layer is made of natural type I collagen and a polymer. The dense layer is composed of several sub-dense layers, wherein the content of the polymer decreases sequentially and the content of natural type I collagen increases sequentially in the sub-dense layers. The sub-dense layer with a high content of natural type I collagen is connected to the loose layer by a crosslinking agent. In this invention, the dense layer is made by electrospinning a mixture of natural type I collagen and a polymer. The dense layer is a gradient spun layer, where the content of the polymer gradually decreases from high to low, while the content of collagen gradually increases from low to high. This dense layer configuration of the invention can gradually release collagen while ensuring the membrane's isolation function and mechanical properties, thereby continuously promoting regeneration.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical materials, and in particular to a double-layer guided tissue regeneration membrane and a preparation method thereof. Background Art

[0002] Dental implants have become an important means of treating tooth defects and missing teeth. However, the lack of bone mass caused by natural atrophy of the alveolar ridge, trauma, periodontal disease and other reasons reduces the success rate of implant restoration. The alveolar ridge of some patients will be too narrow or too low, causing local depression and perforation on the side of the implant, which further leads to the failure of the dental implant surgery. The application of guided tissue regeneration technology (GTR) can effectively solve the problems of insufficient bone mass and alveolar ridge defects.

[0003] Dental guided tissue regeneration technology is a new technology developed in the late 1980s and early 1990s. It is a method frequently used in oral clinical practice to treat bone defects and obtain periodontal tissue regeneration. It can cover the bone defect with an oral repair regeneration membrane to form a barrier to achieve a sealing effect, relieve the pressure of the covering tissue, and protect the blood clot to form a good bone formation space. It can block the fibroblasts and connective tissue that affect bone formation, allowing precursor osteoblasts with growth potential to enter the bone defect area and induce regeneration of the bone repair in the defect area, thereby increasing the success rate of dental implant restoration.

[0004] Tissue engineering scaffold materials are an important factor in tissue engineering construction. The selection of appropriate scaffold materials plays a very important role in the success of regenerative repair strategies. At present, the absorbable oral repair membranes that are widely used at home and abroad are mainly polylactic acid oral repair membranes and collagen oral repair membranes. Collagen membranes that have achieved better results have been most widely used in clinical practice. Collagen-based GTR guided tissue regeneration membranes have good adhesion, good tissue compatibility, low immunogenicity, and can provide effective support for more than 8 weeks, but they are only used as shielding membranes and have poor guiding regeneration ability; in addition, polylactic acid artificial membranes are hydrophobic, have poor absorption of exudate, cannot effectively form blood clots, and increase blood supply and nutrients for the regeneration of damaged tissues, resulting in poor results in inducing bone regeneration.

[0005] In addition, most of the guided tissue regeneration membranes reported in the literature or patents are designed in the form of double-layer or multi-layer membranes to achieve the barrier and regeneration functions of the membrane. However, the guided tissue regeneration membranes of each layer structure are prone to breakage and delamination. The adhesion of each layer of the membrane is not tight, resulting in the separation of the dense layer and the loose layer during use, affecting the use effect. Patent CN115006597A provides an oral repair membrane and a preparation method thereof. The dense layer is prepared by vacuum compounding of multiple layers of membrane-like extracellular matrix. The simple physical method of combination does not provide sufficient firmness between the layers of the membrane, affecting the treatment effect. Patent CN105457107A provides a dual-function oral repair membrane and a preparation method thereof. The repair membrane consists of a loose porous layer and a dense antibacterial layer. The double-layer structure is compounded by two vacuum freeze-drying processes. The combination method is physical combination, and the fit and firmness are poor. It is easy to break and delaminate during use.

[0006] In summary, the existing technology has at least the following problems: the regeneration membrane has a single function, only has a shielding effect, and does not have a good bone induction regeneration function; the membrane has poor absorption and water lock performance, cannot effectively provide nutrients to the bone defect area, and the regeneration effect is not good; and the existing regeneration membranes are mostly physically combined between the layers, with poor firmness, and are prone to stratification and fracture during clinical use, affecting the mechanical stability of the product and resulting in poor use effect. Therefore, it is urgent to develop a composite guided tissue regeneration membrane to solve the above technical problems. Summary of the invention

[0007] The purpose of the present invention is to provide a double-layer guided tissue regeneration membrane and a preparation method thereof to solve the problems existing in the prior art.

[0008] The present invention is achieved through the following technical solutions:

[0009] A double-layer guided tissue regeneration membrane, characterized in that the double-layer guided tissue regeneration membrane comprises: a dense layer and a loose layer;

[0010] The material of the dense layer includes: natural type I collagen and high molecular polymer;

[0011] The dense layer is composed of several stacked sub-dense layers, and the content of the high molecular polymer in the several sub-dense layers decreases successively, and the content of the natural type I collagen increases successively; the sub-dense layer with a high content of natural type I collagen is connected to the loose layer through a cross-linking agent.

[0012] Specifically, the dense layer in the present invention is made of natural type I collagen and high molecular polymer mixed electrostatic spinning, the dense layer is a gradient spinning layer, the content of high molecular polymer gradually decreases from high to low, and the content of collagen gradually increases from low to high; the dense layer setting of the present invention can gradually release collagen under the premise of ensuring the isolation function and mechanical properties of the membrane layer, and continuously achieve the purpose of promoting regeneration. The side with more collagen content is connected to the loose layer, and in the process of collagen degradation over time, cells can gradually penetrate from the loose layer to the dense layer.

[0013] Further, a double-layer guided tissue regeneration membrane: the high molecular polymer is selected from any one or more of polylactic acid, polyglycolic acid, polylactic-co-glycolic acid (PLGA), polycaprolactone, polylactic-co-caprolactone copolymer, and polyethylene glycol copolymer, preferably polylactic acid.

[0014] Furthermore, a double-layer guided tissue regeneration membrane: the thickness of the dense layer is 0.1 to 1.0 mm; the dense layer is composed of three sub-dense layers stacked in sequence, namely the first sub-dense layer, the second sub-dense layer and the third sub-dense layer; the content of the high molecular polymer in the first sub-dense layer, the second sub-dense layer and the third sub-dense layer decreases in sequence, but the content of natural type I collagen increases in sequence; the third sub-dense layer is connected to the loose layer by a cross-linking agent.

[0015] Furthermore, a double-layer guided tissue regeneration membrane: the cross-linking agent is aldehyde-modified hyaluronic acid (OHA).

[0016] Specifically, the aldehyde-modified hyaluronic acid used in the present invention not only cross-links the collagen of each layer, but also serves the purpose of connection, connecting the loose layer and the dense layer, forming a stable chemical bond at the interface, and ensuring the mechanical properties of the membrane during use. The present invention uses aldehyde-modified hyaluronic acid as a cross-linking agent to connect the two layers of the membrane in a wet state, providing the best reaction conditions; on the one hand, the collagen of each layer is cross-linked; on the other hand, by cross-linking the collagen of the dense layer and the loose layer, a connecting effect is played at the interface of the double-layer membrane.

[0017] Furthermore, a double-layer guided tissue regeneration membrane: the preparation method of the aldehyde-modified hyaluronic acid comprises the following steps:

[0018] (1) mixing a hyaluronic acid aqueous solution and a sodium periodate aqueous solution for reaction;

[0019] (2) Ethylene glycol is then added to terminate the reaction, and the mixture is dialyzed and freeze-dried to obtain aldehyde-modified hyaluronic acid.

[0020] Preferably, the aldehyde-modified hyaluronic acid cross-linking agent used in the present invention, on the one hand, cross-links the collagen of each layer, enhances the mechanical properties of the membrane, and prolongs the degradation time; on the other hand, by cross-linking the collagen of the dense layer and the loose layer, it plays a connecting role at the interface of the double-layer membrane, making the double-layer membrane more firmly bonded and ensuring that the layers do not crack during use.

[0021] Further, a double-layer guided tissue regeneration membrane: the preparation method of the aldehyde-modified hyaluronic acid:

[0022] Step (1) adding the sodium periodate aqueous solution dropwise to the hyaluronic acid aqueous solution, and reacting at 20 to 40° C. in the dark for 3 to 6 hours;

[0023] Wherein: the concentration of the hyaluronic acid aqueous solution is 1.0-5.0wt%; the concentration of the sodium periodate aqueous solution is 50-150mg / ml; the volume ratio of the hyaluronic acid aqueous solution to the sodium periodate aqueous solution is 1:(0.5-1.0).

[0024] Furthermore, a double-layer guided tissue regeneration membrane: the thickness of the loose layer is 0.1-1.0 mm.

[0025] A method for preparing a double-layer guided tissue regeneration membrane, characterized in that the method is used to prepare the above-mentioned double-layer guided tissue regeneration membrane, and the method comprises the following steps:

[0026] 1. Preparation of dense layer:

[0027] S1. Dissolving natural type I collagen and high molecular polymer in different proportions in an organic solvent according to the content of the material in each sub-dense layer to obtain a plurality of spinning solutions;

[0028] S2, electrospinning the spinning solutions respectively to form sub-dense layers in sequence until a complete dense layer is obtained;

[0029] 2. Preparation of loose layer:

[0030] S1. Mix the collagen solution with the buffer solution, and then add calcium phosphate salt and stir evenly to obtain a mixed solution;

[0031] S2, allowing the mixed solution to stand and cool to obtain a collagen hydrogel;

[0032] S3, extruding the collagen hydrogel into shape, and drying to obtain the loose layer;

[0033] 3. Preparation of double-layer guided tissue regeneration membrane:

[0034] S1, dissolving the cross-linking agent to obtain a cross-linking solution;

[0035] S2, immersing the loose layer in the cross-linking liquid;

[0036] S3, attaching the impregnated loose layer to the dense layer for reaction;

[0037] S4. After the reaction, freeze-drying is performed to obtain a double-layer guided tissue regeneration membrane.

[0038] Specifically, the present invention provides a double-layer guided tissue regeneration membrane prepared by combining electrospinning technology with self-assembly technology.

[0039] Wherein: the dense layer is made of natural type I collagen and high molecular polymer mixed electrospinning, the dense layer is a gradient spinning layer, the content of high molecular polymer gradually decreases from high to low, and the content of collagen gradually increases from low to high; the dense layer of the present invention can gradually release collagen under the premise of ensuring isolation function and mechanical properties, and continuously achieve the purpose of promoting regeneration. The side with more collagen content is connected to the loose layer, and in the process of collagen degradation over time, cells can gradually penetrate from the loose layer to the dense layer.

[0040] The loose layer is prepared by self-assembly technology. Collagen in the body spontaneously assembles into orderly collagen fibers and collagen fiber bundles, and then forms a macroscopic three-dimensional network structure, which is the structural framework of many biological tissues. The spontaneously formed fiber structure is highly consistent with the matrix tissue. By controlling the concentration of collagen solution and phosphate solution to regulate the density of the fiber, a loose layer structure that is more conducive to cell adhesion, crawling and growth is prepared. Natural bone component calcium phosphate is added to the collagen solution, which is evenly dispersed in the three-dimensional network structure during the self-assembly process, providing calcium and phosphorus elements for the formation of new bone.

[0041] Specifically, the preparation structure of the loose layer highly simulates human bone tissue, which is more conducive to bone growth; the gradient membrane is used as the dense layer, and the electrospinning technology is used to control the pores of the membrane. Collagen is gradually released while ensuring the isolation function and mechanical properties, thereby continuously achieving the purpose of promoting regeneration. The cells gradually penetrate and grow from the loose layer to the dense layer.

[0042] Further, a method for preparing a double-layer guided tissue regeneration membrane: 1. Preparation of a dense layer:

[0043] The mass ratio of natural type I collagen to high molecular weight polymer in step S1 is (1-9): (1-9);

[0044] The mass ratio of the total mass of the natural type I collagen and the high molecular weight polymer described in step S1 to the mass ratio of the organic solvent is 1:(5-20);

[0045] The organic solvent is selected from hexafluoroisopropanol, trifluoroacetic acid, chloroform, dichloromethane and tetrahydrofuran.

[0046] Further, a method for preparing a double-layer guided tissue regeneration membrane: 1. Preparation of a dense layer:

[0047] Wherein: Step S2 includes the following specific steps:

[0048] S2-1, respectively loading several portions of spinning solution into electrospinning syringes; then adjusting the propulsion speed of the electrospinning syringe to 2-6 ml / h, the voltage of the electrospinning machine to 10-20 KV, and the receiving distance of the receiving device to 8-20 cm;

[0049] S2-2, electrospinning is performed in sequence according to the stacking order of each sub-dense layer until a complete dense layer is obtained.

[0050] Further, a method for preparing a double-layer guided tissue regeneration membrane: 2. Preparation of the loose layer:

[0051] Step S1, mixing the collagen solution and the buffer solution in a volume ratio of 1:(2-5), and then adding calcium phosphate salt and stirring evenly to obtain a mixed solution;

[0052] Wherein: the collagen solution is a collagen acetic acid solution with a concentration of 1.0 to 20.0 mg / ml; the buffer is a phosphate buffer;

[0053] The mass ratio of the calcium phosphate salt to the collagen is 1:(2-10).

[0054] Further, a method for preparing a double-layer guided tissue regeneration membrane: 2. Preparation of a loose layer: The calcium phosphate salt described in step S1 is selected from one or more of hydroxyapatite, tricalcium phosphate, tetracalcium phosphate, octacalcium phosphate, calcium hydrogen phosphate, calcium dihydrogen phosphate, and fluoroapatite; the particle size of the calcium phosphate salt is 20 to 200 μm. The preferred calcium phosphate salt is hydroxyapatite.

[0055] Preferably, the smaller the particle size of the calcium phosphate salt, the more conducive it is for osteoblasts to secrete bone matrix, thereby promoting bone formation.

[0056] Further, a method for preparing a double-layer guided tissue regeneration membrane: 2. Preparation of the loose layer:

[0057] Step S2, adjusting the pH of the mixed solution to 5-8, then standing at 35-45° C. for 2-4 hours, cooling to room temperature and standing for 6-10 hours to obtain collagen hydrogel.

[0058] Furthermore, a method for preparing a double-layer guided tissue regeneration membrane is provided: 3. Preparation of a double-layer guided tissue regeneration membrane: Step S1, dissolving the cross-linking agent in water to obtain a cross-linking solution with a concentration of 0.1-10.0 wt%.

[0059] Further, a method for preparing a double-layer guided tissue regeneration membrane: 3. Preparation of a double-layer guided tissue regeneration membrane:

[0060] Step S2, immersing the loose layer in the cross-linking solution for 1 to 5 minutes.

[0061] Further, a method for preparing a double-layer guided tissue regeneration membrane: 3. Preparation of a double-layer guided tissue regeneration membrane:

[0062] The reaction time in step S3 is 10 to 60 minutes.

[0063] Beneficial effects of the present invention:

[0064] (1) The double-layer guided tissue regeneration membrane provided by the present invention uses natural bone component collagen and calcium phosphate as the main raw materials, and adopts a combination of electrospinning and self-assembly technology. The prepared double-layer guided tissue regeneration membrane is made by cross-linking a dense layer and a loose layer. The double-layer membrane is connected by chemical bonds, the combination is more firm, and the mechanical properties are significantly improved; at the same time, the dense layer can play a good barrier role, and the loose layer has good absorption and water locking effects, which solves the problem that the existing synthetic polymer regeneration membrane does not have good liquid absorption performance, cannot effectively lock the seepage blood to form a stable blood clot, and leads to poor induction of bone regeneration effect; the double-layer guided tissue regeneration membrane provided by the present invention is an ideal material for guiding alveolar bone regeneration.

[0065] (2) The double-layer membrane in the present invention is connected by aldehyde-modified hyaluronic acid. After the loose layer is soaked in the aldehyde-modified hyaluronic acid, it is attached to the dense layer in a wet state, and the solution can penetrate into the dense layer. During the static process, not only the non-toxic cross-linking of the collagen in the loose layer and the dense layer is completed, but also a stable connection structure is formed at the interface. Although it is a double-layer membrane, it is actually an integrated structure, and the layers are not easy to crack. The obtained regenerated membrane has a stable structure, good mechanical properties, a long degradation time, and good absorption and permeation performance, which solves the problems of stratification and fracture, poor shielding effect, and unstable implantation in the body caused by loose bonding between the layers of the existing regenerated membrane.

[0066] (3) The present invention optimizes the design of the dense layer structure and controls the material content gradient in the dense layer, thereby ensuring that the dense layer can gradually release the collagen in the layer while having both isolation function and mechanical properties, thereby achieving the purpose of continuously promoting regeneration; the loose layer prepared by the present invention also has the function of facilitating cell adhesion, crawling and growth, which can further promote the improvement of regeneration ability. In summary, the regeneration membrane provided by the present invention solves the problem that the current guided tissue regeneration membrane has a single function, only has a shielding effect, and has poor guided regeneration ability.

[0067] (4) The double-layer guided tissue regeneration membrane of the present invention is mainly composed of natural ingredients such as collagen, hyaluronic acid and hydroxyapatite (calcium phosphate), which provides an optimal environment for bone regeneration. In addition, hydroxyapatite has a certain hardness, which can increase the supporting performance of the membrane.

[0068] (5) Compared with the existing guided tissue regeneration membrane's water absorption function alone, the present invention not only enhances the absorption and permeability through the addition of aldehyde-modified hyaluronic acid, but also increases the water-locking property, which can effectively lock the bleeding and avoid the loss of the composite regeneration factor in the blood, which is more conducive to bone regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0070] Figure 1 This is an infrared detection image of the aldehyde-modified hyaluronic acid prepared in Example 1 of the present invention;

[0071] Figure 2 The water absorption ratio diagram of the double-layer tissue regeneration membranes obtained in Examples 1-2 of the present invention and Comparative Examples 1-2 is shown. DETAILED DESCRIPTION

[0072] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0073] In the description of the present invention, it should be understood that the terms "upper", "lower", "left", "right", "top", "bottom", etc., indicating the position or positional relationship, are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Moreover, the terms "first", "second", etc. are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchangeable where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein.

[0074] Example 1

[0075] A double-layer guided tissue regeneration membrane, characterized in that the double-layer guided tissue regeneration membrane comprises: a dense layer with a thickness of 0.3 mm and a loose layer with a thickness of 0.2 mm;

[0076] The material of the dense layer includes: natural type I collagen and polylactic acid (polymer); the dense layer is composed of three sub-dense layers of the same thickness stacked in sequence, namely the first sub-dense layer, the second sub-dense layer and the third sub-dense layer;

[0077] The content of polylactic acid in the first sub-dense layer, the second sub-dense layer and the third sub-dense layer decreases successively, but the content of natural type I collagen increases successively; the third sub-dense layer is connected to the loose layer through aldehyde-modified hyaluronic acid (cross-linking agent).

[0078] The method for preparing a double-layer guided tissue regeneration membrane provided in the above embodiment 1 comprises the following specific steps:

[0079] 1. Preparation of dense layer:

[0080] S1, preparing a first spinning solution by mixing natural type I collagen and polylactic acid in a mass ratio of 1:9, and the total mass of natural type I collagen and polylactic acid and hexafluoroisopropanol in a mass ratio of 1:10, and using the first spinning solution for electrospinning to form a first sub-dense layer;

[0081] The mass ratio of natural type I collagen to polylactic acid is 1:1, and the total mass of natural type I collagen and polylactic acid to hexafluoroisopropanol is 1:10 to prepare a second spinning solution, and the second spinning solution is used for electrospinning to form a second sub-dense layer;

[0082] The third spinning solution is prepared by mixing the natural type I collagen and polylactic acid in a mass ratio of 9:1 and the total mass of the natural type I collagen and polylactic acid with hexafluoroisopropanol in a mass ratio of 1:10. The third spinning solution is used for electrospinning to form a third sub-dense layer.

[0083] S2. Step S2 includes the following specific steps:

[0084] S2-1, respectively loading the first spinning solution, the second spinning solution and the third spinning solution into the electrospinning syringe; then adjusting the propulsion speed of the electrospinning syringe to 4 ml / h, the voltage of the electrospinning machine to 15 KV, and the receiving distance of the receiving device to 15 cm;

[0085] S2-2, performing electrospinning in sequence according to the stacking order of the first sub-dense layer, the second sub-dense layer and the third sub-dense layer until a complete dense layer is obtained;

[0086] 2. Preparation of loose layer:

[0087] S1. Dissolve collagen in acetic acid solution and stir evenly to obtain a collagen solution with a concentration of 10.0 mg / ml; mix the collagen solution and phosphate buffer solution at a volume ratio of 1:3, and then add hydroxyapatite (calcium phosphate) with an average particle size of 30 μm and stir evenly to obtain a mixed solution; wherein: the mass ratio of hydroxyapatite to collagen is 1:3;

[0088] S2, adjusting the pH of the mixed solution to 7.4, then standing at 37° C. for 3 hours, cooling to room temperature and standing for 8 hours to obtain a collagen hydrogel;

[0089] S3, placing the collagen hydrogel into a mold, squeezing it flat, and vacuum drying it for 24 hours to obtain a loose layer with a thickness of 0.2 mm and a pore size of about 15 μm;

[0090] 3. Preparation of double-layer guided tissue regeneration membrane:

[0091] S1. Add a sodium periodate aqueous solution with a volume ratio of 0.5:1 and a concentration of 80 mg / ml to a hyaluronic acid aqueous solution with a concentration of 1.0 wt%, and react at 35° C. in the dark for 5 hours; then add ethylene glycol to terminate the reaction, dialyze, and freeze-dry to obtain aldehyded hyaluronic acid; dissolve the aldehyded hyaluronic acid (cross-linking agent) in water to obtain an aldehyded hyaluronic acid solution with a concentration of 0.5 wt%;

[0092] S2, immersing the loose layer in the aldehyde-modified hyaluronic acid solution for 5 minutes;

[0093] S3, drain the excess solution from the impregnated loose layer, attach it to the dense layer in a wet state, that is, attach it to the third sub-dense layer, and let it stand for 50 minutes to fully react;

[0094] S4. After the reaction, the membrane was freeze-dried for 24 hours to obtain a double-layer guided tissue regeneration membrane.

[0095] Example 2

[0096] A double-layer guided tissue regeneration membrane, characterized in that the double-layer guided tissue regeneration membrane comprises: a dense layer with a thickness of 0.2 mm and a loose layer with a thickness of 0.3 mm;

[0097] The material of the dense layer includes: natural type I collagen and polycaprolactone (high molecular polymer); the dense layer is composed of three sub-dense layers with the same thickness stacked in sequence, namely the first sub-dense layer, the second sub-dense layer and the third sub-dense layer;

[0098] The content of polycaprolactone in the first subdense layer, the second subdense layer and the third subdense layer decreases successively, but the content of natural type I collagen increases successively; the third subdense layer is connected to the loose layer through aldehyde-modified hyaluronic acid (cross-linking agent).

[0099] The method for preparing a double-layer guided tissue regeneration membrane provided in the above-mentioned embodiment 2 comprises the following specific steps:

[0100] 1. Preparation of dense layer:

[0101] S1, mixing natural type I collagen and polycaprolactone in a mass ratio of 1:8, and natural type I collagen and polycaprolactone and trifluoroacetic acid in a mass ratio of 1:5 to prepare a first spinning solution, and the first spinning solution is used for electrospinning to form a first sub-dense layer;

[0102] The second spinning solution is prepared by mixing the natural type I collagen and polycaprolactone in a mass ratio of 1:1 and the natural type I collagen and polycaprolactone and trifluoroacetic acid in a mass ratio of 1:5, and the second spinning solution is used for electrospinning to form a second sub-dense layer;

[0103] The third spinning solution is prepared by mixing the natural type I collagen and polycaprolactone in a mass ratio of 8:1 and the natural type I collagen and polycaprolactone and trifluoroacetic acid in a mass ratio of 1:5. The third spinning solution is used for electrospinning to form a third sub-dense layer.

[0104] S2. Step S2 includes the following specific steps:

[0105] S2-1, respectively loading the first spinning solution, the second spinning solution and the third spinning solution into the electrospinning syringe; then adjusting the propulsion speed of the electrospinning syringe to 5 ml / h, the voltage of the electrospinning machine to 18 KV, and the receiving distance of the receiving device to 13 cm;

[0106] S2-2, performing electrospinning in sequence according to the stacking order of the first sub-dense layer, the second sub-dense layer and the third sub-dense layer until a complete dense layer is obtained;

[0107] 2. Preparation of loose layer:

[0108] S1. Dissolve collagen in acetic acid solution and stir evenly to obtain a collagen solution with a concentration of 20.0 mg / ml; mix the collagen solution and phosphate buffer solution at a volume ratio of 1:5, and then add tricalcium phosphate (calcium phosphate) with an average particle size of 35 μm and stir evenly to obtain a mixed solution; wherein the mass ratio of tricalcium phosphate to collagen is 1:5;

[0109] S2, adjusting the pH of the mixed solution to 7.4, then standing at 45° C. for 2 hours, cooling to room temperature and standing for 6 hours to obtain a collagen hydrogel;

[0110] S3, placing the collagen hydrogel into a mold, squeezing it flat, and vacuum drying it for 24 hours to obtain a loose layer with a thickness of 0.3 mm;

[0111] 3. Preparation of double-layer guided tissue regeneration membrane:

[0112] S1. A sodium periodate aqueous solution with a volume ratio of 0.8:1 and a concentration of 100 mg / ml is added dropwise to a hyaluronic acid aqueous solution with a concentration of 2.0 wt%, and the mixture is reacted at 40° C. in the dark for 6 hours; ethylene glycol is then added to terminate the reaction, and the mixture is dialyzed and freeze-dried to obtain aldehyded hyaluronic acid; the aldehyded hyaluronic acid (cross-linking agent) is dissolved in water to obtain an aldehyded hyaluronic acid solution with a concentration of 1.0 wt%;

[0113] S2, immersing the loose layer in the aldehyde-modified hyaluronic acid solution for 3 minutes;

[0114] S3, drain the excess solution from the impregnated loose layer, attach it to the dense layer in a wet state, that is, attach it to the third sub-dense layer, and let it stand for 60 minutes for full reaction;

[0115] S4. After the reaction, the membrane was freeze-dried for 24 hours to obtain a double-layer guided tissue regeneration membrane.

[0116] Example 3

[0117] A double-layer guided tissue regeneration membrane, characterized in that the double-layer guided tissue regeneration membrane comprises: a dense layer with a thickness of 1.0 mm and a loose layer with a thickness of 1.0 mm;

[0118] The material of the dense layer includes: natural type I collagen and polylactic acid glycolic acid copolymer (polymer); the dense layer is composed of three sub-dense layers with the same thickness stacked in sequence, namely the first sub-dense layer, the second sub-dense layer and the third sub-dense layer;

[0119] The content of polylactic acid-glycolic acid copolymer in the first sub-dense layer, the second sub-dense layer and the third sub-dense layer decreases successively, but the content of natural type I collagen increases successively; the third sub-dense layer is connected to the loose layer through aldehyde-modified hyaluronic acid (cross-linking agent).

[0120] The method for preparing a double-layer guided tissue regeneration membrane provided in the above-mentioned embodiment 3 comprises the following specific steps:

[0121] 1. Preparation of dense layer:

[0122] S1, mixing natural type I collagen and poly(lactic acid-co-glycolic acid) copolymer in a mass ratio of 1:2, and natural type I collagen and polycaprolactone and tetrahydrofuran in a mass ratio of 1:20 to prepare a first spinning solution, and the first spinning solution is used for electrospinning to form a first sub-dense layer;

[0123] The second spinning solution is prepared by mixing the natural type I collagen and poly(lactic-co-glycolic acid) copolymer in a mass ratio of 1:1, and the natural type I collagen and polycaprolactone and tetrahydrofuran in a mass ratio of 1:20. The second spinning solution is used for electrospinning to form a second sub-dense layer.

[0124] The third spinning solution is prepared by mixing the natural type I collagen and poly(lactic-co-glycolic acid) copolymer in a mass ratio of 2:1, and the natural type I collagen and polycaprolactone and tetrahydrofuran in a mass ratio of 1:20. The third spinning solution is used for electrospinning to form a third sub-dense layer.

[0125] S2. Step S2 includes the following specific steps:

[0126] S2-1, respectively loading the first spinning solution, the second spinning solution and the third spinning solution into the electrospinning syringe; then adjusting the propulsion speed of the electrospinning syringe to 3 ml / h, the voltage of the electrospinning machine to 10 KV, and the receiving distance of the receiving device to 8 cm;

[0127] S2-2, performing electrospinning in sequence according to the stacking order of the first sub-dense layer, the second sub-dense layer and the third sub-dense layer until a complete dense layer is obtained;

[0128] 2. Preparation of loose layer:

[0129] S1. Dissolve collagen in acetic acid solution and stir evenly to obtain a collagen solution with a concentration of 3.0 mg / ml; mix the collagen solution and phosphate buffer solution at a volume ratio of 1:2, and then add calcium dihydrogen phosphate (calcium phosphate) with an average particle size of 20 μm and stir evenly to obtain a mixed solution; wherein: the mass ratio of calcium dihydrogen phosphate to collagen is 1:10;

[0130] S2, adjusting the pH of the mixed solution to 5.5, then standing at 35° C. for 4 hours, cooling to room temperature and standing for 10 hours to obtain a collagen hydrogel;

[0131] S3, placing the collagen hydrogel into a mold, squeezing it flat, and vacuum drying it for 24 hours to obtain a loose layer with a thickness of 1.0 mm;

[0132] 3. Preparation of double-layer guided tissue regeneration membrane:

[0133] S1. Add a sodium periodate aqueous solution with a volume ratio of 1:1 and a concentration of 50 mg / ml to a hyaluronic acid aqueous solution with a concentration of 5.0 wt%, and react at 20° C. in the dark for 3 hours; then add ethylene glycol to terminate the reaction, dialyze, and freeze-dry to obtain aldehyded hyaluronic acid; dissolve the aldehyded hyaluronic acid (cross-linking agent) in water to obtain an aldehyded hyaluronic acid solution with a concentration of 10.0 wt%;

[0134] S2, immersing the loose layer in the aldehyde-modified hyaluronic acid solution for 4 minutes;

[0135] S3, drain the excess solution from the impregnated loose layer, attach it to the dense layer in a wet state, that is, attach it to the third sub-dense layer, and let it stand for 20 minutes for full reaction;

[0136] S4. After the reaction, the membrane was freeze-dried for 24 hours to obtain a double-layer guided tissue regeneration membrane.

[0137] Comparative Example 1

[0138] The preparation process of the dense layer in Comparative Example 1 is the same as that in Example 1; the preparation process of the loose layer in Comparative Example 1 is the same as that in Example 1. The difference between Comparative Example 1 and Example 1 is that the preparation of the double-layer tissue regeneration membrane is different; the preparation steps of the double-layer tissue regeneration membrane in Comparative Example 1 are as follows:

[0139] ① Prepare a 0.5wt% glutaraldehyde solution, the solvent of which is an 80wt% ethanol aqueous solution;

[0140] ② Immerse the dried loose layer and dense layer in the prepared glutaraldehyde solution respectively for 5 minutes;

[0141] ③Take out the loose layer and the dense layer, drain the excess solution, and let them stand for 60 minutes to fully react;

[0142] ④ Rinse the loose layer and the dense layer three times with deionized water, and then immerse the loose layer and the dense layer in a 0.5wt% lysine solution at 4°C for 2 hours to seal the glutaraldehyde in the loose layer and the dense layer;

[0143] ⑤ After soaking, rinse thoroughly with deionized water three times, put the two layers of membrane into a mold and press them by gravity, and freeze-dry for 24 hours to obtain a double-layer tissue regeneration membrane with a thickness of 0.5 mm.

[0144] Comparative Example 2

[0145] The difference between Comparative Example 2 and Example 1 is that hyaluronic acid is used in Comparative Example 2, which is different from the aldehyde-modified hyaluronic acid in Example 1; the rest of Comparative Example 2 is the same as Example 1.

[0146] test:

[0147] (1) Cytotoxicity test: Cytotoxicity test was performed on the double-layer tissue regeneration membranes prepared in Examples 1 to 3 and Comparative Examples 1 to 2: L929 cells were selected, and serum-containing culture medium was added to each group of test samples, extracted at 37°C for 24 hours, and the extract was added to the cells and cultured for 24 hours. Cytotoxicity test was performed using the MTT method (n=3). The test results are shown in Table 1. Table 1 shows the cytotoxicity test results of the double-layer tissue regeneration membranes prepared in Examples 1 to 3 and Comparative Examples 1 to 2

[0148]

[0149]

[0150] It can be seen from Table 1 that the cell viability of the sample groups of Examples 1 to 3 and Comparative Example 2 were 95.25%, 94.49%, 95.03% and 94.87%, respectively, indicating that the prepared double-layer guided tissue regeneration membrane was non-cytotoxic (i.e., aldehyde-modified hyaluronic acid was used as a cross-linking agent, which was safe and non-toxic); while the cross-linking agent of Comparative Example 1 was glutaraldehyde. Although a step of treating residual glutaraldehyde was added in the process, a small amount of glutaraldehyde still remained, resulting in a lower cell viability than that of other sample groups. It can be seen that residual glutaraldehyde has a certain effect on cells.

[0151] (2) Infrared detection: Using the KBr tableting method, a small amount of hyaluronic acid (HA) and the aldehyded hyaluronic acid (OHA) prepared in Example 1 of the present invention were placed in a mortar, KBr powder was added, and tablets were pressed. The HA and OHA were measured using a Fourier transform infrared spectrometer at a wavelength of 400 to 4000 cm -1 The samples were characterized within the range of Figure 1 As shown, from Figure 1 It can be seen that the infrared spectra of OHA and HA are similar, but at 1732cm-1 A new characteristic peak appears at , which corresponds to the stretching vibration of -C=O in the aldehyde group and has the characteristics of OHA, indicating that the method of the present invention can successfully prepare aldehyded hyaluronic acid (OHA).

[0152] (3) In vitro degradation and tensile strength test:

[0153] ① In vitro degradation: Weigh the samples obtained in Examples 1 to 3 and Comparative Examples 1 to 2, and record the weight as W0. Aseptically immerse each sample in a PBS buffer solution at 37°C and let it stand. Take samples at 4, 8, and 12 weeks, and weigh the weight after vacuum drying as W1. Calculate the degradation rate according to the following formula. The degradation calculation results are shown in Table 2:

[0154]

[0155] Table 2 shows the degradation rate results

[0156]

[0157]

[0158] It can be seen from Table 2 that the double-layer guided regeneration membrane prepared by the present invention has a slow degradation rate and can gradually release collagen to continuously achieve the effect of promoting regeneration.

[0159] ②Tensile strength test: The samples after degradation for 0, 4, 8, and 12 weeks were cut into samples with a length of 20 mm and a width of 10 mm, and then the tensile strength (MPa) was measured at a speed of 1 mm / min using a universal testing machine. The test results are shown in Table 3:

[0160] Table 3 shows the tensile strength test results.

[0161] Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 0 Weeks 12.22 12.36 19.64 11.39 5.44 4 weeks 9.70 9.27 16.58 5.63 3.97 8 weeks 7.99 7.29 12.73 3.81 1.71 12 weeks 4.93 4.95 7.90 1.99 -

[0162] Note: “-” means the sample is not formed and cannot be weighed or tested

[0163] It can be seen from the results of Table 3 that as time increases, each group of samples begins to degrade; because the double-layer membranes of Examples 1 to 3 and Comparative Example 1 have been cross-linked, when the degradation reaches 12 weeks, the degradation rates are 68.39%, 69.97%, 58.89% and 70.93%, respectively, and the basic structure can still be maintained, and it can be seen that the cross-linking properties of the regenerated membranes of the aldehyde-modified hyaluronic acid and the commonly used cross-linking agent glutaraldehyde used in the present invention have no significant difference; Comparative Example 2 is not cross-linked, and when the degradation reaches 12 weeks, no membrane structure is observed, and the degradation is basically complete. In addition, the tensile strength of the samples of Examples 1 to 3 is still about 5 to 8MPa after the degradation reaches 12 weeks, which has good mechanical strength and can meet clinical use. Comparative Example 1 has no chemical bonding process at the double-layer interface, which causes the double-layer membrane to be not firmly combined, cracking during the immersion process, affecting the tensile strength, and when Comparative Example 2 reaches 12 weeks, the mechanical strength cannot be detected.

[0164] (4) Test of absorption and permeation performance: The absorption and permeation performance of the membrane was tested by immersing samples of Examples 1 to 2 and Comparative Examples 1 to 2 of the same weight in PBS solution at 37° C. for 6 hours, and comparing the weight W before immersion with the weight W after immersion. 0h And the weight after 6 hours of immersion W 6h The water absorption rate is calculated by the following formula: The test results are as follows Figure 2 As shown:

[0165] The water absorption rate can be used to intuitively judge the ability of the membrane material to retain moisture and absorb water. Examples 1 to 2 can absorb more than fifty times their own weight in water. Since Comparative Example 1 does not have a formaldehyde-modified hyaluronic acid component, the water absorption rate is greatly reduced and it can only absorb 17.9 times its own weight in water. Although hyaluronic acid is added to Comparative Example 2, there is no cross-linking process, and the hyaluronic acid dissolves and loses. As time goes by, the loss will be more serious, and it cannot play the role of absorbing and locking water.

[0166] The above are preferred embodiments of the present invention and are only used to explain the present invention, not to limit the present invention. Any obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A double-layer guided tissue regeneration membrane, characterized in that: The double-layer guided tissue regeneration membrane comprises: a dense layer and a loose layer; The material of the dense layer includes: natural type I collagen and high molecular polymer; The dense layer is composed of a plurality of stacked sub-dense layers, and the content of the high molecular weight polymer in the plurality of sub-dense layers decreases successively, and the content of the natural type I collagen increases successively; The sub-dense layer with a high content of natural type I collagen is connected to the loose layer through a cross-linking agent; the cross-linking agent is aldehyde-modified hyaluronic acid; The loose layer is obtained by extruding and drying a collagen hydrogel, and the collagen hydrogel is obtained by mixing a collagen solution with a buffer solution and a calcium phosphate salt, and then standing and cooling; The volume ratio of the collagen solution to the buffer solution is 1:(2-5), the mass ratio of the calcium phosphate salt to the collagen is 1:(2-10), the pH of the system is adjusted to 5-8 after mixing, and the standing temperature is 35-45°C.

2. A double-layer guided tissue regeneration membrane according to claim 1, characterized in that: The high molecular polymer is selected from any one or more of polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, polycaprolactone, polylactic acid-caprolactone copolymer, and polyethylene glycol copolymer.

3. A double-layer guided tissue regeneration membrane according to claim 1, characterized in that: The thickness of the dense layer is 0.1 to 1.0 mm; The dense layer is composed of three sub-dense layers stacked in sequence, namely a first sub-dense layer, a second sub-dense layer and a third sub-dense layer; The content of the high molecular weight polymer in the first sub-dense layer, the second sub-dense layer and the third sub-dense layer decreases in sequence, but the content of natural type I collagen increases in sequence; The third sub-dense layer is connected to the loose layer through a cross-linking agent.

4. The double-layer guided tissue regeneration membrane according to claim 1, characterized in that: The preparation method of the aldehyded hyaluronic acid comprises the following steps: (1) mixing a hyaluronic acid aqueous solution and a sodium periodate aqueous solution for reaction; (2) Ethylene glycol is then added to terminate the reaction, and the mixture is dialyzed and freeze-dried to obtain aldehyde-modified hyaluronic acid.

5. The double-layer guided tissue regeneration membrane according to claim 4, characterized in that: The preparation method of the aldehyde-modified hyaluronic acid: Step (1) adding the sodium periodate aqueous solution dropwise to the hyaluronic acid aqueous solution, and reacting at 20 to 40° C. in the dark for 3 to 6 hours; Wherein: the concentration of the hyaluronic acid aqueous solution is 1.0-5.0wt%; the concentration of the sodium periodate aqueous solution is 50-150mg / ml; the volume ratio of the hyaluronic acid aqueous solution to the sodium periodate aqueous solution is 1:(0.5-1).

6. The double-layer guided tissue regeneration membrane according to claim 1, characterized in that: The thickness of the loose layer is 0.1-1.0 mm.

7. A method for preparing a double-layer guided tissue regeneration membrane, characterized in that: The method is used to prepare the double-layer guided tissue regeneration membrane according to any one of claims 1 to 6, and the method comprises the following steps:

1. Preparation of dense layer: S1. Dissolving natural type I collagen and high molecular polymer in different proportions in an organic solvent according to the content of the material in each sub-dense layer to obtain a plurality of spinning solutions; S2, electrospinning the spinning solutions respectively to form sub-dense layers in sequence until a complete dense layer is obtained; 2. Preparation of loose layer: S1. Mix the collagen solution with the buffer solution, and then add calcium phosphate salt and stir evenly to obtain a mixed solution; S2, allowing the mixed solution to stand and cool to obtain a collagen hydrogel; S3, extruding the collagen hydrogel into shape, and drying to obtain the loose layer; 3. Preparation of double-layer guided tissue regeneration membrane: S1, dissolving the cross-linking agent to obtain a cross-linking solution; S2, immersing the loose layer in the cross-linking liquid; S3, attaching the impregnated loose layer to the dense layer for reaction; S4. After the reaction, freeze-drying is performed to obtain a double-layer guided tissue regeneration membrane.

8. The method for preparing a double-layer guided tissue regeneration membrane according to claim 7, characterized in that:

1. Preparation of dense layer: The mass ratio of natural type I collagen to high molecular weight polymer described in step S1 is (1-9): (1-9); The mass ratio of the total mass of the natural type I collagen and the high molecular weight polymer described in step S1 to the mass ratio of the organic solvent is 1:(5-20); The organic solvent is selected from hexafluoroisopropanol, trifluoroacetic acid, chloroform, dichloromethane and tetrahydrofuran.

9. The method for preparing a double-layer guided tissue regeneration membrane according to claim 7, characterized in that:

1. Preparation of dense layer: Wherein: Step S2 includes the following specific steps: S2-1, respectively loading several portions of spinning solution into electrospinning syringes; then adjusting the propulsion speed of the electrospinning syringe to 2-6 ml / h, the voltage of the electrospinning machine to 10-20 KV, and the receiving distance of the receiving device to 8-20 cm; S2-2, electrospinning is performed in sequence according to the stacking order of each sub-dense layer until a complete dense layer is obtained.

10. The method for preparing a double-layer guided tissue regeneration membrane according to claim 7, characterized in that:

2. Preparation of loose layer: The collagen solution in step S1 is a collagen acetic acid solution with a concentration of 1.0 to 20.0 mg / ml; the buffer is a phosphate buffer.

11. The method for preparing a double-layer guided tissue regeneration membrane according to claim 7, characterized in that:

2. Preparation of loose layer: The calcium phosphate salt described in step S1 is selected from one or more of hydroxyapatite, tricalcium phosphate, tetracalcium phosphate, octacalcium phosphate, calcium hydrogen phosphate, calcium dihydrogen phosphate, and fluoroapatite; The particle size of the calcium phosphate salt is 20 to 200 μm.

12. The method for preparing a double-layer guided tissue regeneration membrane according to claim 7, characterized in that:

2. Preparation of loose layer: Step S2, adjusting the pH of the mixed solution to 5-8, then standing at 35-45° C. for 2-4 hours, cooling to room temperature and standing for 6-10 hours to obtain collagen hydrogel.

13. The method for preparing a double-layer guided tissue regeneration membrane according to claim 7, characterized in that:

3. Preparation of double-layer guided tissue regeneration membrane: Step S1, dissolving the cross-linking agent in water to obtain a cross-linking solution with a concentration of 0.1 to 10.0 wt%.

14. The method for preparing a double-layer guided tissue regeneration membrane according to claim 7, characterized in that:

3. Preparation of double-layer guided tissue regeneration membrane: Step S2, immersing the loose layer in the cross-linking solution for 1 to 5 minutes.

15. The method for preparing a double-layer guided tissue regeneration membrane according to claim 7, characterized in that:

3. Preparation of double-layer guided tissue regeneration membrane: The reaction time in step S3 is 10 to 60 minutes.

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