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

Abstract

The present application relates to the technical field of biological materials, in particular to a double-layer guided tissue regeneration membrane and a preparation method thereof, so as to improve the comprehensive performance of the double-layer guided tissue regeneration membrane. In the preparation, collagen type I is dissolved in acid to prepare a collagen type I solution; then, an alkali is added to adjust the pH value, so as to prepare collagen type I fibers; then, the collagen type I fibers are processed and molded, and gradient freeze-drying is performed, so as to prepare a collagen type I membrane; then, the collagen type I membrane is crosslinked with a crosslinking agent, so as to prepare the double-layer guided tissue regeneration membrane; wherein, the concentration of the collagen type I solution is 0.5-1.5%, and the pH value is 7-9. The double-layer guided tissue regeneration membrane prepared by the above preparation method is composed of a dense layer and a loose layer formed on the surface of the dense layer, the thickness of the dense layer is 0.2-0.8 mm, and the thickness of the loose layer is 0.4-0.8 mm. The tensile strength is greater than or equal to 28 N, the suture strength is greater than or equal to 4.7 N, and the porosity is greater than or equal to 65.8%, which effectively solves the problems of insufficient mechanical performance and low biological safety of the existing double-layer guided tissue regeneration membrane. The preparation process is simple, green and large-scale.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and in particular to a bilayer guided tissue regeneration membrane and its preparation method. Background Technology

[0002] Guided tissue regeneration membranes are membranes that selectively guide cells to attach and proliferate at damaged sites, thereby achieving tissue repair, through mechanisms such as mechanical barriers. The key to tissue repair and regeneration lies in the development and application of functional regenerative materials. Currently, commonly used regeneration membranes can be categorized into non-degradable and biodegradable membranes based on their raw materials. Biodegradable membranes are relatively rare on the market. Xenogeneic biomaterial-based tissue regeneration membranes are currently the most widely used tissue regeneration membranes both domestically and internationally, including Bio-Gide collagen membranes, Giteri medical collagen membranes, and Hai'ao oral repair membranes. However, these membranes all suffer from drawbacks such as poor mechanical properties, short degradation times, and unsatisfactory guided tissue regeneration characteristics.

[0003] There are some reports both domestically and internationally of using collagen components to construct guided regeneration membranes. Patent CN1586637A uses type I collagen as the main raw material to prepare a bilayer composite guided tissue regeneration membrane. The dense layer is made of collagen through pressing, and the loose layer is composed of collagen and hyaluronic acid or its sodium salt. The bilayer composite membrane is prepared into a finished product through cross-linking agent, cleaning, and freeze-drying. However, this method of preparing the guided membrane still requires mechanical pressure molding, and is not a direct process molding, making the process complex.

[0004] Patent US2004 / 0214991A1 describes a collagen and glycosaminoglycan complex formed by impregnating a collagen membrane in a mixed solution of type I collagen and glycosaminoglycans, freezing, vacuum freeze-drying, extruding, and cross-linking, which is used to guide tissue regeneration.

[0005] Patent CN102716517A describes the use of collagen or sodium hyaluronate as raw materials to produce collagen membranes or collagen / sodium hyaluronate composite membranes through traditional processes such as dissolution and freeze-drying. Although the immunogenicity of the raw materials is removed, the mechanical properties of the materials are poor, and sutures cannot be used to fuse with the tissue during implantation.

[0006] Patent CN105214141A provides a three-dimensional composite material for tendon and ligament repair. Different materials are stacked during preparation. However, this physical combination has poor adhesion and firmness, and is prone to breakage and delamination during use. The layers are not tightly bonded, resulting in the separation of the dense layer and the loose layer during use.

[0007] Therefore, finding a tissue regeneration membrane that is simple to process, has good mechanical properties, high porosity, and good biocompatibility has become an urgent technical problem to be solved in this field. Summary of the Invention

[0008] The purpose of this invention is to provide a double-layer guided tissue regeneration membrane and its preparation method, thereby improving the overall performance of double-layer guided tissue regeneration membranes in the prior art.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of the present invention provides a method for preparing a bilayer guided tissue regeneration membrane, comprising the following steps: (1) Type I collagen solution was prepared by acid dissolving Type I collagen; (2) Add alkali to the type I collagen solution to adjust the pH value and obtain type I collagen fibers; (3) The type I collagen fibers are processed and shaped, and then freeze-dried in a gradient manner to obtain a type I collagen membrane; (4) The type I collagen membrane is cross-linked with a cross-linking agent to obtain the double-layer guided tissue regeneration membrane; in step (1), the concentration of the type I collagen solution is 0.5~1.5%; in step (2), the pH value is 7~9; In step (3), the gradient freeze drying includes a low-temperature freezing stage, a first drying stage, a second drying stage, and a third drying stage; During the low-temperature freezing stage, the freezing temperature is -40 to -20°C, and the freezing time is 600 to 750 minutes. In the first drying stage, the drying temperature is -5~0℃ and the drying time is 600~750min; In the second drying stage, the drying temperature is 5~15℃ and the drying time is 600~750min; In the third drying stage, the drying temperature is 20~25℃ and the drying time is 600~750min.

[0010] Further, in step (1), during acid dissolution, the acid is a mixture of one or more of acetic acid, lactic acid and sodium citrate; the concentration of the acid solution is 0.4~0.8 mol / L.

[0011] Further, in step (2), the alkali is ammonia or sodium hydroxide, and the concentration of the alkali solution is 0.2%~0.5%.

[0012] Further, in step (4), the crosslinking agent is a mixture of one or more selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, glutaraldehyde and genipin, and the concentration of the crosslinking agent is 1~3%.

[0013] Further, in step (1), the type I collagen is extracted from bovine Achilles tendon, and the specific extraction method includes the following steps: S1: After slicing, removing impurities and defatting the bovine Achilles tendon in sequence, pre-treated bovine Achilles tendon slices are obtained; S2: The bovine Achilles tendon slices are subjected to acid dissolution, enzymatic hydrolysis, salting out, and freeze-drying in sequence to extract the type I collagen.

[0014] Further, in step S1, the thickness of the bovine Achilles tendon slice is 2-5 mm; during impurity removal, the bovine Achilles tendon slice is soaked in sodium chloride solution and then rinsed with water; during degreasing, the bovine Achilles tendon slice after impurity removal is soaked in degreasing solution and then rinsed with water.

[0015] Furthermore, during impurity removal, the concentration of the sodium chloride solution is 8%~12%, and the soaking time is 18~28 hours; during degreasing, the degreasing solution is an isopropanol solution, and the soaking time is 18~28 hours.

[0016] Further, in step S2, during enzymatic hydrolysis, the enzyme is a mixture of one or more selected from pepsin, papain, chymotrypsin, and trypsin; the concentration of the enzyme is 0.08~0.8%.

[0017] A second aspect of the present invention provides a double-layer guided tissue regeneration membrane, which is prepared by the above-described method for preparing a double-layer guided tissue regeneration membrane. The double-layer guided tissue regeneration membrane consists of a dense layer and a loose layer formed on the surface of the dense layer. The thickness of the dense layer is 0.2 to 0.8 mm, and the thickness of the loose layer is 0.4 to 0.8 mm.

[0018] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art: This invention utilizes an acid-dissolution-alkali precipitation method to reconstruct collagen fibers, then processes and shapes the reconstructed collagen fibers, followed by cross-linking with a cross-linking agent to obtain a bilayer guided tissue regeneration membrane. Through direct molding, the tensile strength, suture strength, and porosity of the bilayer guided tissue regeneration membrane are improved, achieving a tensile strength ≥28N, a suture strength ≥4.7N, and a porosity ≥65.8%, effectively addressing the problems of insufficient mechanical properties and low biosafety in existing bilayer guided tissue regeneration membranes. The preparation process is simple, environmentally friendly, and suitable for large-scale production. Attached Figure Description

[0019] The following sections will describe some specific embodiments of the invention in detail by way of example and not limitation, with reference to the accompanying drawings. The same reference numerals in the drawings denote the same or similar parts or portions. Those skilled in the art should understand that these drawings are not necessarily drawn to scale. In the drawings: Figure 1 This is a schematic diagram of the structure of the double-layer guided tissue regeneration membrane provided in this invention; Figure 2 This is a flowchart illustrating the preparation process of the bilayer guided tissue regeneration membrane provided in this invention. Figure 3 This is a graph showing the results of cell proliferation activity detection for Example 1 and Comparative Example 1 provided in this invention; Figure 4 This is a dense layer scanning electron microscope image of the double-layer guided tissue regeneration membrane prepared in Example 1 of this invention; Figure 5 This is a scanning electron microscope image of the loose layer of the double-layer guided tissue regeneration membrane prepared in Example 1 of this invention; Figure 6 This is a cross-sectional scanning electron microscope image of the bilayer guided tissue regeneration membrane prepared in Example 1 of this invention.

[0020] The reference numerals in the attached figures are as follows: 1. Dense layer; 2. Loose layer. Detailed Implementation

[0021] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. Example 1

[0023] This embodiment provides a bilayer guided tissue regeneration membrane and its preparation method, see reference. Figure 2 It includes the following steps: (1) Extraction of type I collagen Take frozen beef Achilles tendon and slice it into 3mm thick slices using a slicer; then place the slices in a 10% sodium chloride solution and soak them at 4°C for 24 hours, then rinse them repeatedly with cold pure water 3 times; then place them in isopropanol for defatting and stirring for 24 hours, and then rinse them repeatedly with cold pure water. The defatted sheets were placed in a 0.5 mol / L acetic acid solution, and 0.1% pepsin was added. After enzymatic hydrolysis at room temperature for 72 hours, 1% hydrogen peroxide was added, and the hydrolysis was stopped after stirring for 3 hours. After the hydrolysis was completed, 10% sodium chloride was added to the solution (at a ratio of 1:1 between the collagen solution and the sodium chloride solution) for salting out, and then the solution was repeatedly rinsed with low-temperature pure water. The collagen was homogenized in a 0.5 mol / L acetic acid solution for 20 minutes using a homogenizer. The resulting solution was then poured into a medical stainless steel freezing pan and frozen at -30°C for 600 minutes. The temperature was then raised to 23°C and dried for 3000 minutes to produce the collagen sponge.

[0024] (2) Preparation of a double-layer guided tissue regeneration membrane Take 3.75g of collagen sponge, dissolve it in 500mL of 0.5mol / L acetic acid solution to prepare a collagen solution with a concentration of 0.75%. Homogenize it for 8 min, then let it stand to remove bubbles to obtain the collagen solution. Add 0.4% sodium hydroxide to the above collagen solution to adjust the pH of the solution to 8. At this time, the collagen self-assembles and reconstructs to form white clump-like fibers. The filtered and reconstructed collagen fibers were wound onto a 1.5cm diameter polytetrafluoroethylene rod using a top-mounted electric stirrer at 100 rpm to obtain a collagen membrane. After the winding is completed, the above collagen membrane is subjected to gradient freeze drying. First, it is frozen at -20℃ for 700 min; then the temperature is raised to 0℃ and dried for 700 min; then the temperature is raised to 10℃ and dried for 700 min; finally the temperature is raised to 25℃ and dried for 700 min. EDC was added to 1000 mL of 50% ethanol solution to prepare a 2% EDC alcohol solution. The collagen membrane was then placed in the EDC alcohol solution for cross-linking for 24 h, washed with pure water, and finally lyophilized for storage. This yielded a bilayer tissue regeneration guiding membrane. (See [reference needed]). Figure 1 The thickness of the dense layer 1 in the regenerated membrane is 0.5 mm, and the pore size is between 0 and 5 µm; the thickness of the loose layer 2 is 0.5 mm, and the pore size is between 80 and 315 µm. Example 2

[0025] This embodiment provides a double-layer guided tissue regeneration membrane and its preparation method. The only difference between this embodiment and Embodiment 1 is that the collagen solution concentration in step (2) is 1.5%. Example 3

[0026] This embodiment provides a double-layer guided tissue regeneration membrane and its preparation method. The only difference between this embodiment and Embodiment 1 is that the collagen solution concentration in step (2) is 0.5%. Example 4

[0027] This embodiment provides a bilayer guided tissue regeneration membrane and its preparation method. The only difference between this embodiment and Embodiment 1 is that the gradient freeze-drying process for the collagen membrane in step (2) is different. First, the membrane is frozen at -20°C for 700 min; then heated to 0°C and dried for 700 min; then heated to 12°C and dried for 700 min; finally heated to 25°C and dried for 700 min. The thickness of the dense layer 1 in the bilayer guided tissue regeneration membrane prepared by this embodiment is 0.7 mm, and the pore size is between 0 and 5 µm; the thickness of the loose layer 2 is 0.3 mm, and the pore size is between 75 and 280 µm. Example 5

[0028] This embodiment provides a bilayer guided tissue regeneration membrane and its preparation method. The only difference between this embodiment and Embodiment 1 is that the gradient freeze-drying process for the collagen membrane in step (2) is different. First, the membrane is frozen at -20°C for 700 min; then heated to 0°C and dried for 700 min; then heated to 8°C and dried for 700 min; finally heated to 25°C and dried for 700 min. The thickness of the dense layer 1 in the bilayer guided tissue regeneration membrane prepared by this embodiment is 0.4 mm, and the pore size is between 0 and 5 µm; the thickness of the loose layer 2 is 0.6 mm, and the pore size is between 70 and 300 µm. Example 6

[0029] This embodiment provides a bilayer guided tissue regeneration membrane and its preparation method. The only difference between this embodiment and Embodiment 1 is that the gradient freeze-drying process for the collagen membrane in step (2) is different. First, the membrane is frozen at -20°C for 700 min; then heated to 0°C and dried for 700 min; then heated to 5°C and dried for 700 min; finally heated to 25°C and dried for 700 min. The thickness of the dense layer 1 in the bilayer guided tissue regeneration membrane prepared by this embodiment is 0.2 mm, and the pore size is between 0 and 5 µm; the thickness of the loose layer 2 is 0.8 mm, and the pore size is between 50 and 300 µm.

[0030] Comparative Example 1 This embodiment provides a collagen membrane and its preparation method. The only difference from Embodiment 1 is that no processing and forming device is used to receive the membrane. After freeze-drying, a single-layer collagen membrane with a thickness of 1 mm and a pore size between 80 and 350 µm is obtained.

[0031] In this invention, the collagen membranes prepared in the above embodiments and comparative examples were subjected to performance tests and characterization. The characterization results are as follows: (1) Porosity detection Table 1: Porosity test results for each sample

[0032] As shown in the table above, the porosity of the process of this invention is relatively high, providing more growth space for cells.

[0033] (2) Cell proliferation activity Cell proliferation activity was tested in Example 1 and Comparative Example 1: L929 cells in logarithmic growth phase were digested and diluted to a density of 2*102. 4 Cells / mL: After sterilization, collagen membranes were cut and soaked in complete culture medium for 24 hours, then transferred to 24-well plates. 1 mL of cell suspension was added to each well, and the plates were incubated at 37°C in a 5% CO2 incubator. Cell viability was measured using CCK-8 (10%) solution on days 1, 3, 5, and 7. Results are shown below. Figure 3 As shown in the figure, analysis of the data reveals that the OD value of the material group in Example 1 exhibited a continuous and significant increasing trend from day 1 to day 7. Although the OD value of the material group in Comparative Example 1 also increased with the cultivation time, its growth rate and the final OD value level were both lower than those of the material group in Example 1.

[0034] The results directly demonstrate that, compared with Comparative Example 1 (traditional single-layer membrane), the bilayer guided tissue regeneration membrane prepared in Example 1 of the present invention has superior cell compatibility, can provide a more suitable three-dimensional microenvironment for cell growth, and can significantly support and promote cell adhesion and proliferation.

[0035] (3) Tensile strength test The sample film was trimmed into rectangular specimens of 150mm × 20mm. The specimens were placed on a testing machine for tensile strength testing. The experimental conditions were: clamp spacing of 100mm and clamp separation speed of 50mm / min. The maximum tensile strength was measured on a mechanical testing instrument, and the results are shown in the table below: Table 2: Comparison of Mechanical Test Data for Each Sample

[0036] As can be seen from the table above, the comparative example and the embodiment have different processing methods. The membrane prepared by the process method of the embodiment of the present invention has significantly stronger biomechanical strength than the comparative example.

[0037] (4) Test of suture tensile strength The test conditions for suture tensile strength were as follows: clamp spacing 100 mm, clamp separation speed 100 mm / min, initial strain rate 0.5 mm / min. 3-0# non-absorbable suture was used, with a double strand threaded through the center of the sample; each strand was 75 mm long.

[0038] Table 3: Tensile Strength of Sutures in Examples and Comparative Cases

[0039] In addition, the bilayer guided tissue regeneration membrane prepared in Example 1 was characterized by SEM morphology. Figure 4 It can be seen that the dense layer 1 of the collagen membrane in Example 1 is relatively smooth and dense, with almost no micropores, and the average pore size is 0~5µm; from Figure 5 As can be seen, this is the rough porous surface of Example 1, with an average pore size of 80~315µm. From... Figure 6 As can be seen, the double-layer structure is clearly visible, with the dense layer 1 and the loose layer 2 tightly bonded together.

[0040] Comparison of data from Comparative Example 1 and the embodiments of the present invention demonstrates that while traditional freeze-drying processes can achieve extremely high porosity, they inevitably lead to a loss of mechanical strength, failing to meet clinical needs. The present invention, through a unique acid-dissolution and alkali-extraction reconstruction of collagen fibers combined with a winding molding process, achieves a synergistic balance between high porosity and high strength in the tissue regeneration membrane. Furthermore, by adjusting gradient freeze-drying parameters, the performance profile of the product can be customized within a certain range (e.g., Example 6 emphasizes high porosity, Example 1 has the best overall performance, and Example 4 emphasizes mechanical support), thereby meeting diverse clinical needs.

[0041] In summary, this invention develops a bilayer guided tissue regeneration membrane and its preparation method. Collagen fibers are reconstructed using an acid-dissolution-alkali precipitation method, and then the reconstructed collagen fibers are processed and shaped. Subsequently, they are cross-linked with a cross-linking agent to obtain a bilayer guided tissue regeneration membrane. This results in a unique three-dimensional structure with high porosity but also good fiber interweaving and orientation, thereby achieving a qualitative leap in mechanical properties while maintaining high porosity.

[0042] By employing a direct molding process, the tensile strength, suture strength, and porosity of the bilayer guided tissue regeneration membrane are improved, resulting in a tensile strength ≥28N, a suture strength ≥4.7N, and a porosity ≥65.8%. This effectively addresses the issues of insufficient mechanical properties and low biosafety in existing bilayer guided tissue regeneration membranes. The preparation process is simple, environmentally friendly, and suitable for large-scale production.

[0043] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a double-layer guided tissue regeneration membrane, characterized in that, Includes the following steps: (1) Type I collagen solution was prepared by acid dissolving Type I collagen; (2) Add alkali to the type I collagen solution to adjust the pH value and obtain type I collagen fibers; (3) The type I collagen fibers are wound into shape and then freeze-dried in a gradient to obtain a type I collagen membrane; (4) The type I collagen membrane is cross-linked with a cross-linking agent to obtain the double-layer guided tissue regeneration membrane; in step (1), the concentration of the type I collagen solution is 0.5~1.5%; in step (2), the pH value is 7~9; In step (3), the gradient freeze drying includes a low-temperature freezing stage, a first drying stage, a second drying stage, and a third drying stage; During the low-temperature freezing stage, the freezing temperature is -40 to -20°C, and the freezing time is 600 to 750 minutes. In the first drying stage, the drying temperature is -5~0℃ and the drying time is 600~750min; In the second drying stage, the drying temperature is 5~15℃ and the drying time is 600~750min; In the third drying stage, the drying temperature is 20~25℃ and the drying time is 600~750min.

2. The method for preparing a double-layer guided tissue regeneration membrane according to claim 1, characterized in that, In step (1), during acid dissolution, the acid is a mixture of one or more of acetic acid, lactic acid and sodium citrate; the concentration of the acid solution is 0.4~0.8 mol / L.

3. The method for preparing a double-layer guided tissue regeneration membrane according to claim 1, characterized in that, In step (2), the alkali is ammonia or sodium hydroxide, and the concentration of the alkali solution is 0.2% to 0.5%.

4. The method for preparing a double-layer guided tissue regeneration membrane according to claim 1, characterized in that, In step (4), the crosslinking agent is a mixture of one or more selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, glutaraldehyde and genipin, and the concentration of the crosslinking agent is 1~3%.

5. The method for preparing a double-layer guided tissue regeneration membrane according to claim 1, characterized in that, In step (1), the type I collagen is extracted from bovine Achilles tendon. The specific extraction method includes the following steps: S1: After slicing, removing impurities and defatting the bovine Achilles tendon in sequence, pre-treated bovine Achilles tendon slices are obtained; S2: The bovine Achilles tendon slices are subjected to acid dissolution, enzymatic hydrolysis, salting out, and freeze-drying in sequence to extract the type I collagen.

6. The method for preparing a double-layer guided tissue regeneration membrane according to claim 5, characterized in that, In step S1, the thickness of the bovine Achilles tendon slices is 2-5 mm; during impurity removal, the bovine Achilles tendon slices are soaked in sodium chloride solution and then rinsed with water; during degreasing, the bovine Achilles tendon slices after impurity removal are soaked in a degreasing solution and then rinsed with water.

7. A method for preparing a double-layer guided tissue regeneration membrane according to claim 6, characterized in that, During the impurity removal process, the concentration of the sodium chloride solution is 8%~12%, and the soaking time is 18~28 hours; during the degreasing process, the degreasing solution is an isopropanol solution, and the soaking time is 18~28 hours.

8. The method for preparing a double-layer guided tissue regeneration membrane according to claim 5, characterized in that, In step S2, during enzymatic hydrolysis, the enzyme is a mixture of one or more of pepsin, papain, chymotrypsin, and trypsin; the concentration of the enzyme is 0.08~0.8%.

9. A double-layer guided tissue regeneration membrane prepared by the method described in any one of claims 1 to 8, characterized in that, It consists of a dense layer and a loose layer formed on the surface of the dense layer, wherein the thickness of the dense layer is 0.2~0.8mm and the thickness of the loose layer is 0.4~0.8mm.

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