Composite anti-adhesion membrane as well as preparation method and application thereof

Abstract

The invention relates to a composite anti-adhesion membrane as well as a preparation method and application thereof. The composite anti-adhesion membrane comprises a decellularized visceral membrane layer and a collagen-chondroitin sulfate composite sponge layer arranged on the decellularized visceral membrane layer. The invention provides the composite anti-adhesion membrane with a double-layer structure comprising the decellularized visceral membrane layer and the collagen-chondroitin sulfate composite sponge layer, the two layers can be tightly combined, the front and back surfaces have proper pore sizes, porosity, tensile strength and softness are relatively high, the degradation time is relatively long, and the anti-adhesion membrane has a good anti-adhesion effect. The application requirements of postoperative tissue anti-adhesion materials can be met.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a composite anti-adhesion membrane, its preparation method, and its application. Background Technology

[0002] Postoperative tissue adhesions are a common problem in the field of surgery, especially in anatomical areas where blood vessels, tendons, and nerves are densely distributed (such as the carpal tunnel and ankle joint). Adhesions between blood vessels, tendons, nerves, and surrounding tissues can lead to serious consequences. For example, vascular adhesions may lead to hemodynamic disturbances (vascular stenosis / thrombosis), tendon adhesions may lead to gliding dysfunction, and nerve adhesions may lead to chronic pain or entrapment syndrome.

[0003] Existing anti-adhesion materials mainly include synthetic polymer membranes and natural polymer membranes. Among them, synthetic polymer membranes, such as polylactic acid membranes, have excessively high elastic modulus, which may compress blood vessels / nerves, and the acidic degradation products may trigger inflammatory reactions. Natural polymer membranes, such as pure collagen membranes, have defects such as excessively rapid degradation or insufficient mechanical strength. The pore size of pure collagen membranes is uneven (50~200μm) and cannot block fibroblast migration. On the other hand, simple decellularized matrix has a dense overall pore size, which is not conducive to the problem of close adhesion with tissues.

[0004] LU503769B1 discloses a collagen fiber composite membrane for repairing nerve damage. The collagen fiber composite membrane includes a three-layer structure: an anti-adhesion layer, an elastic connecting layer, and a regeneration promoting layer. The layers are connected by chemical cross-linking. The anti-adhesion layer is cast using type I collagen, the intermediate layer is methacryloyl gelatin, and the regeneration promoting layer is electrospun type I collagen. However, the mechanical strength of this product is generally not high, making it difficult to provide good protection for exposed tendons, nerves, etc.

[0005] Therefore, it is necessary to provide a suitable anti-adhesion membrane that meets the functional requirements of anti-adhesion, protection, and regeneration promotion. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a composite anti-adhesion membrane, its preparation method, and its applications. The composite anti-adhesion membrane provided by this invention has two layers that can be tightly bonded together. Both sides have appropriately sized pores and porosity, high tensile strength and flexibility, and a long degradation time. It is suitable for surgical procedures requiring simultaneous protection of blood vessels, tendons, and nerves (such as carpal tunnel release and tendon-nerve anastomosis).

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a composite anti-adhesion membrane, the composite anti-adhesion membrane comprising a decellularized visceral membrane layer and a collagen-chondroitin sulfate composite sponge layer disposed on the decellularized visceral membrane layer.

[0009] The composite anti-adhesion membrane provided by this invention comprises a two-layer structure consisting of a decellularized visceral membrane layer and a collagen-chondroitin sulfate composite sponge layer. The combination of collagen and chondroitin sulfate reduces the viscosity of the collagen solution, resulting in a more porous structure after freeze-drying. The two layers are tightly bonded together, with suitable pore sizes and porosity on both sides. It exhibits high tensile strength and flexibility, and a long degradation time, thus meeting the functional requirements of anti-adhesion, protection, and regeneration promotion.

[0010] Preferably, the decellularized visceral membrane layer is derived from the peritoneum, pericardium, small intestinal mucosa, or myofascia of pigs, cattle, or horses.

[0011] Preferably, the thickness of the decellularized visceral membrane layer is 0.3~1.2mm (e.g., 0.3mm, 0.5mm, 0.8mm, 1mm, 1.2mm, etc.), and the in vivo degradation cycle is not less than 30 days (e.g., 30 days, 31 days, 32 days, 35 days, 40 days, etc.).

[0012] Preferably, the surface pore size of the decellularized visceral membrane layer is 0.5~10μm (e.g., it can be 0.5μm, 1μm, 2μm, 5μm, 8μm, 10μm, etc.).

[0013] Preferably, in the collagen-chondroitin sulfate composite sponge layer, the collagen content is 90-98 wt% (e.g., 90 wt%, 92 wt%, 94 wt%, 96 wt%, 98 wt%, etc.), and the chondroitin sulfate content is 2-10 wt% (e.g., 2%, 2.5%, 2.6%, 5%, 6%, 8%, 10%, etc.).

[0014] Preferably, the thickness of the collagen-chondroitin sulfate composite sponge layer is 0.5~1.5mm (e.g., 0.5mm, 0.8mm, 1mm, 1.2mm, 1.5mm, etc.), and the in vivo degradation cycle is not less than 30 days (e.g., 30 days, 31 days, 32 days, 35 days, 40 days, etc.).

[0015] Preferably, the surface pore size of the collagen-chondroitin sulfate composite sponge layer is 30~300μm (e.g., it can be 30μm, 50μm, 100μm, 150μm, 200μm, 300μm, etc.).

[0016] In a second aspect, the present invention provides a method for preparing the composite anti-adhesion film according to the first aspect, the method comprising the following steps:

[0017] (1) The decellularized visceral membrane is acidified or alkalized to obtain a pretreated membrane layer;

[0018] (2) Roughen the rough surface of the pretreated film and let it stand to obtain a surface gelled film.

[0019] (3) The collagen raw material, chondroitin sulfate, crosslinking agent and solution are mixed and crosslinked, and then injected onto the surface of the surface gelled film layer. After freeze drying and heat treatment, the composite anti-adhesion film is obtained.

[0020] In this invention, although the pore size of the untreated decellularized visceral membrane is small, it is still greater than 10 μm, allowing fibroblasts to grow in. After acidification or alkalization treatment, the pore size can be further reduced.

[0021] In this invention, the rough surface of the decellularized matrix is ​​scraped to remove the fibrous layer and gelled to increase its contact area with the collagen solution, making the double layer bond tighter. At the same time, it prevents the cross-linking agent from penetrating into the decellularized visceral membrane, which would harden if chemical cross-linking occurred.

[0022] This invention employs a method of first attaching the visceral membrane of the cell and then perfusing it with collagen solution to create a composite layer. The components of the two layers can come into close contact, ensuring a tight bond between them.

[0023] Preferably, the acidification or alkalization treatment in step (1) includes soaking.

[0024] Preferably, in step (1), the decellularized visceral membrane is acidified, and the acid used in the acidification process is an aqueous solution of acetic acid.

[0025] Preferably, the concentration of the acetic acid aqueous solution is 0.01~0.1 mol / L (e.g., 0.01 mol / L, 0.02 mol / L, 0.05 mol / L, 0.08 mol / L, 0.1 mol / L, etc.), and the acidification treatment time is 3 min or more (e.g., 3 min, 5 min, 10 min, 15 min, etc.), preferably 3~12 min.

[0026] Preferably, the roughening process in step (2) includes scraping fibers onto the rough surface of the pretreated film using a rigid material.

[0027] Preferably, the rigid material includes a steel ruler.

[0028] Preferably, the settling time in step (2) is 1 to 20 minutes (for example, it can be 1 minute, 3 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, etc.).

[0029] Preferably, the collagen raw material in step (3) includes purified collagen and / or decellularized visceral membrane.

[0030] Preferably, when the collagen raw material in step (3) is decellularized visceral membrane, the process further includes homogenizing the decellularized visceral membrane and the dissolving solution.

[0031] Preferably, the crosslinking agent in step (3) includes glutaraldehyde.

[0032] Preferably, the solution in step (3) includes an aqueous solution of acetic acid, preferably an aqueous solution of acetic acid with a concentration of 0.01~0.1 mol / L (e.g., 0.01 mol / L, 0.02 mol / L, 0.05 mol / L, 0.08 mol / L, 0.1 mol / L, etc.).

[0033] Preferably, the amounts of collagen raw material, chondroitin sulfate and crosslinking agent in step (3) are 0.3~0.8wt% (e.g., 0.3wt%, 0.4wt%, 0.5wt%, 0.6wt%, 0.8wt%, etc.), 0.006~0.09wt% (e.g., 0.006wt%, 0.01wt%, 0.02wt%, 0.05wt%, 0.07wt%, 0.09wt%, etc.) and 0.01~0.1wt% (e.g., 0.01wt%, 0.02wt%, 0.05wt%, 0.08wt%, 0.1wt%, etc.) of the solution, respectively.

[0034] Preferably, the temperature of the heating treatment in step (3) is 100~110℃ (e.g., 100℃, 102℃, 105℃, 108℃, 110℃, etc.), and the time is 0.5~12h (e.g., 0.5h, 2h, 4h, 8h, 12h, etc.).

[0035] Preferably, the heat treatment is performed under vacuum.

[0036] Preferably, the heat treatment further includes washing and sterilization.

[0037] Thirdly, the present invention provides an application of the composite anti-adhesion film according to the first aspect in the preparation of anti-adhesion materials.

[0038] Compared with the prior art, the present invention has at least the following beneficial effects:

[0039] This invention provides a composite anti-adhesion membrane with a double-layer structure comprising a decellularized visceral membrane layer and a collagen-chondroitin sulfate composite sponge layer. The two layers can be tightly bonded together, and both sides have appropriately sized pores and porosity. It has high tensile strength and softness, and a long degradation time, which can meet the application requirements of postoperative tissue anti-adhesion materials. Attached Figure Description

[0040] Figure 1This is a scanning electron microscope image of a longitudinal section of the composite anti-adhesion film provided in Example 3.

[0041] Figure 2 This is a scanning electron microscope image of the front side of the composite anti-adhesion film provided in Example 3.

[0042] Figure 3 This is a scanning electron microscope image of the back side of the composite anti-adhesion film provided in Example 3.

[0043] Figure 4 This is a comparison chart of the sag test results of the composite anti-adhesion films provided in Examples 3 and 6. Detailed Implementation

[0044] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0045] Example 1

[0046] This embodiment provides a method for preparing a composite anti-adhesion film, including the following steps:

[0047] (1) The porcine peritoneum was soaked in 0.01 mol / L acetic acid aqueous solution for 10 min to obtain a pretreated membrane;

[0048] (2) Lay the pretreated film flat on the mold with the rough side facing away from the mold. Use a steel ruler to scrape out fibers on the rough side for roughening treatment. Let it stand for 10 minutes to allow the surface to gel, and obtain a surface gelled film.

[0049] (3) Add porcine peritoneum to 0.01 mol / L acetic acid aqueous solution, add chondroitin sulfate, and then homogenize to prepare a slurry with porcine peritoneum content of 0.7 wt% and chondroitin sulfate content of 0.09 wt%. Add 0.1 wt% glutaraldehyde, stir for 1 h, then degas, pour into the surface of the surface gelled membrane, freeze in an ultra-low temperature freezer, and then freeze-dry. Treat the freeze-dried sample at 100℃ vacuum high temperature for 0.5 h, then wash with water, soak in phosphate buffer, and irradiate to sterilize to obtain a composite anti-adhesion membrane.

[0050] Example 2

[0051] This embodiment provides a method for preparing a composite anti-adhesion film, including the following steps:

[0052] (1) The porcine peritoneum was soaked in 0.1 mol / L acetic acid aqueous solution for 3 min to obtain a pretreated membrane;

[0053] (2) Lay the pretreated film flat on the mold with the rough side facing away from the mold. Use a steel ruler to scrape out fibers on the rough side for roughening treatment. Let it stand for 2 minutes to allow the surface to gel, and obtain a surface gelled film.

[0054] (3) Prepare a 0.5wt% purified collagen solution with 0.03mol / L acetic acid aqueous solution, add 0.05wt% chondroitin sulfate and 0.05wt% glutaraldehyde, stir for 0.5h, then degas, pour into the surface of the surface gelled membrane, put it in an ultra-low temperature freezer to freeze, then freeze-dry, treat the freeze-dried sample at 110℃ vacuum high temperature for 12h, then wash with water, soak in phosphate buffer, and irradiate to sterilize to obtain a composite anti-adhesion membrane.

[0055] Example 3

[0056] This embodiment provides a method for preparing a composite anti-adhesion film, including the following steps:

[0057] (1) The porcine peritoneum was soaked in 0.03 mol / L acetic acid aqueous solution for 5 min to obtain a pretreated membrane;

[0058] (2) Lay the pretreated film flat on the mold with the rough side facing away from the mold. Use a steel ruler to scrape out fibers on the rough side for roughening treatment. Let it stand for 6 minutes to allow the surface to gel, and obtain a surface gelled film.

[0059] (3) Prepare a 0.6wt% purified collagen solution with 0.03mol / L acetic acid aqueous solution, add 0.006wt% chondroitin sulfate and 0.01wt% glutaraldehyde, stir for 0.5h, then degas, pour into the surface of the surface gelled membrane, put it in an ultra-low temperature freezer to freeze, and then freeze-dry. The freeze-dried sample is vacuum-treated at 105℃ for 2h, then washed with water, and soaked in phosphate buffer for irradiation sterilization to obtain a composite anti-adhesion membrane.

[0060] Example 4

[0061] This embodiment provides a method for preparing a composite anti-adhesion film. The only difference between this embodiment and Embodiment 3 is that water immersion is used in step (1), while the other steps are the same as in Embodiment 3.

[0062] Results: The pores of the visceral membrane were 5~30μm, and the surface did not gel after the roughening treatment in step (2).

[0063] Example 5

[0064] This embodiment provides a method for preparing a composite anti-adhesion film. The only difference between this embodiment and Embodiment 3 is that the roughening treatment in step (2) is not performed. The rest is the same as in Embodiment 3.

[0065] Results: The visceral membrane and collagen sponge layer did not bond tightly after freeze-drying and were prone to delamination.

[0066] Example 6

[0067] This embodiment provides a method for preparing a composite anti-adhesion film. The only difference between this method and Example 3 is that in step (3), the vacuum high-temperature treatment time is 24 hours, and the rest is the same as in Example 3.

[0068] Result: High-temperature treatment for 24 hours led to peritoneal dehydration and hardening.

[0069] Comparative Example 1

[0070] This comparative example provides a method for preparing a composite anti-adhesion film. The only difference between this method and Example 3 is that chondroitin sulfate is not added in step (3), while the rest is the same as in Example 3.

[0071] Results: Due to the high viscosity of the collagen solution alone, it was difficult to bind to the gelled peritoneal surface and difficult to perfuse smoothly. The resulting bilayered sample showed varying degrees of stratification after freeze-drying and during water washing.

[0072] Test case

[0073] (1) Aperture: Observed by scanning electron microscope.

[0074] (2) Double layer combination: Observe with the naked eye whether the double layers are tightly bound without separation.

[0075] (3) Drape: Suspend or place a 3cm×2cm material on the tip of a syringe needle with the needle at the center of the material and observe its natural drooping or slipping. Take a picture and measure the angle of curvature on both sides with a protractor. Materials with higher flexibility droop more easily and at smaller angles.

[0076] The samples provided in Examples 1-6 and Comparative Example 1 were tested, and the results are summarized in Table 1. The scanning electron microscope images of Example 3 (longitudinal section, front side, and back side) are shown below. Figure 1-3 As shown in the figure, the comparison diagram of the drape test between Example 3 and Example 6 is as follows. Figure 4 As shown.

[0077] Table 1

[0078]

[0079] in conclusion:

[0080] (1) As can be seen from Examples 1 to 6, the composite anti-adhesion membrane provided by the present invention has a two-layer structure that can be tightly bonded, with appropriate pore size and porosity on both sides, high tensile strength and softness, and long degradation time, which can meet the application requirements of postoperative tissue anti-adhesion materials.

[0081] (2) By comparing Example 1 with Examples 4-6, it can be seen that when no acid treatment is performed, the double layer may separate into layers; when no roughening treatment is performed, the double layer may separate into layers; when the high temperature treatment time is too long, the sample dehydrates and hardens, and the hanging angle increases.

[0082] (3) By comparing Example 1 with Comparative Example 1, it can be seen that when chondroitin sulfate is not added, due to the high viscosity of collagen, the visceral membrane and collagen sponge are prone to occasional delamination.

[0083] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A composite anti-blocking film, characterized by, The composite anti-adhesion membrane comprises a decellularized internal organ membrane layer and a collagen-chondroitin sulfate composite sponge layer arranged on the decellularized internal organ membrane layer.

2. The composite anti-blocking film according to claim 1, wherein The decellularized internal organ membrane layer is derived from a peritoneum, a pericardium, a small intestinal mucosa or a muscle fascia of a pig, a cow or a horse. Preferably, the decellularized internal organ membrane layer has a thickness of 0.3-1.2 mm and an in-vivo degradation period of not less than 30 days. Preferably, the decellularized internal organ membrane layer has a surface pore size of 0.5-10 μm.

3. The composite anti-blocking film according to claim 1 or 2, wherein The collagen-chondroitin sulfate composite sponge layer has a collagen content of 90-98 wt% and a chondroitin sulfate content of 2-10 wt%.

4. The composite anti-blocking film according to any one of claims 1 to 3, wherein, The collagen-chondroitin sulfate composite sponge layer has a thickness of 0.5-1.5 mm and an in-vivo degradation period of not less than 30 days. Preferably, the collagen-chondroitin sulfate composite sponge layer has a surface pore size of 30-300 μm.

5. A method of preparing the composite anti-blocking film according to any one of claims 1-4, characterized in that, The preparation method comprises the following steps: (1) acidizing or alkalizing a decellularized internal organ membrane to obtain a pretreated membrane layer; (2) roughening the rough surface of the pretreated membrane layer and standing to obtain a surface gelatinized membrane layer; (3) mixing collagen raw material, chondroitin sulfate, a cross-linking agent and a dissolving solution, performing cross-linking treatment, then perfusing the surface gelatinized membrane layer with the cross-linking agent, and performing freeze-drying and heating treatment to obtain the composite anti-adhesion membrane.

6. The production method according to claim 5, wherein The acidizing or alkalizing treatment in step (1) can be performed by immersion. Preferably, the acidizing treatment in step (1) is performed using an acetic acid aqueous solution. Preferably, the acetic acid aqueous solution has a concentration of 0.01-0.1 mol / L, and the acidizing treatment is performed for 3 min or more.

7. The production method according to claim 5 or 6, characterized by, The roughening treatment in step (2) can be performed by scratching fibers on the rough surface of the pretreated membrane layer using a rigid material. Preferably, the standing in step (2) is performed for 1-20 min.

8. The method of any one of claims 5-7, wherein, The collagen raw material in step (3) can be purified collagen and / or a decellularized internal organ membrane. Preferably, the cross-linking agent in step (3) is glutaraldehyde. Preferably, the dissolving solution in step (3) is an acetic acid aqueous solution, preferably an acetic acid aqueous solution having a concentration of 0.01-0.1 mol / L. Preferably, the collagen raw material, chondroitin sulfate and cross-linking agent in step (3) are used in an amount of 0.3-0.8 wt%, 0.006-0.09 wt% and 0.01-0.1 wt% of the dissolving solution, respectively.

9. The method of any one of claims 5-8, wherein, The heating treatment in step (3) is performed at a temperature of 100-110 °C for 0.5-12 h.

10. Use of the composite anti-adhesion membrane according to any one of claims 1-4 in the preparation of an anti-adhesion material.

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