Collagen membrane with effect of inducing ordered remodeling of tissues, preparation method therefor, and use thereof

Abstract

A collagen membrane with an effect of inducing ordered remodeling of tissues, a preparation method therefor, and use thereof, relating to the technical field of surgical supply materials. The preparation method for the collagen membrane comprises the following steps: subjecting a fiber membrane to a heat pressing treatment to obtain the collagen membrane with the effect of inducing ordered remodeling of tissues. The fiber membrane is formed by cross-stacked fibers with a diameter of 0.5-15 μm. The fibers comprise collagen and poly(L-lactide-co-caprolactone). The mass ratio of the collagen to the poly(L-lactide-co-caprolactone) is 1:(1.5-2.5). By combining the collagen, which has good biocompatibility and an effect of promoting tissue repair, with PLCL, which is biodegradable and features excellent mechanical properties and good cell adhesion, the collagen membrane not only has an extracellular matrix-like structure, but also exhibits good structural stability, facilitating the regulation of cell behaviors and tissue regeneration, and effectively promoting the growth, adhesion, and proliferation of host cells, thereby inducing ordered remodeling of tissues.

Description

Collagen membrane capable of inducing orderly tissue remodeling, preparation method thereof, and application thereof

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on December 12, 2023, with application number 2023117113735 and invention name “A collagen membrane with the function of inducing orderly tissue remodeling, its preparation method and application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to the technical field of surgical product materials, and more particularly to a collagen membrane capable of inducing orderly tissue remodeling, and a preparation method and application thereof. Background Art

[0003] Tendon and ligament problems are currently the most common musculoskeletal disorders. Tendons and ligaments (T / L), composed of dense connective tissue, transmit force from muscle to bone and bone to bone, respectively, allowing joint movement and force exertion, playing a vital role in normal joint motion and stability. When T / L injuries occur, surgical intervention is primarily used to restore the continuity of the damaged tendon. Specific methods include direct suturing, autologous tendon transplantation, and artificial tendon transplantation. However, these methods have poor long-term clinical efficacy and are associated with numerous adverse reactions, such as re-tear, adhesion formation, scarring, and joint stiffness.

[0004] To address the above-mentioned issues, researchers have developed tissue engineering scaffolds made from tissue remodeling biomaterials. These scaffolds can induce the remodeling, repair, and regeneration of wound tissue by regulating the growth and differentiation of cells surrounding the wound, promoting the regeneration of blood vessels and nerves, and connecting with surrounding tissues. For example, prior art discloses a ligament regeneration scaffold with layer-by-layer induction properties and a method for preparing the same. The ligament regeneration scaffold has a multi-layer composite structure consisting of an overlapping composite of a gradient-degradable micron fiber reinforcement layer and an oriented nanofiber induction layer. The micron fiber reinforcement layer is woven from yarns with different degradation cycles through multi-dimensional, multi-textile molding, while the nanofiber induction layer is electrospinned from polymer materials and bioactive ingredients. By regulating the gradient degradation structure, the micron fiber reinforcement layer is gradiently degraded while maintaining initial mechanical strength, exposing the nanofiber induction layer layer by layer. This improves the infiltration properties of the scaffold's internal tissues and increases the depth of cell penetration within the scaffold. However, the structural stability of the nanofiber induction layer is poor.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to overcome the defects or shortcomings of the existing ligament regeneration scaffolds, such as poor dimensional stability after swelling, and to provide a method for preparing a collagen membrane that can induce orderly tissue remodeling.

[0007] Another object of the present invention is a collagen membrane prepared by the above preparation method that has the function of inducing orderly tissue remodeling.

[0008] Another object of the present invention is the use of the above collagen membrane in preparing tissue regeneration membrane.

[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0010] The present invention provides a method for preparing a collagen membrane capable of inducing orderly tissue remodeling, comprising the following steps:

[0011] The fiber membrane is subjected to heat pressing to obtain a collagen membrane having the function of inducing orderly tissue remodeling; the fiber membrane is formed by cross-layering fibers with an average diameter of 0.5 to 15 μm;

[0012] The fiber comprises collagen and poly (L-lactide-caprolactone), and the mass ratio of the collagen to the poly (L-lactide-caprolactone) is 1:(1.5-2.5).

[0013] The present invention selects fibers of specific diameters to ensure that the fiber diameter in the final collagen membrane is relatively similar to the collagen fiber diameter in the tendon extracellular matrix. The extracellular matrix-like structure formed is beneficial to regulating cell behavior and tissue regeneration, and can provide a favorable regenerative environment for the growth, adhesion and proliferation of host cells, thereby helping to induce orderly tissue remodeling.

[0014] Moreover, the fiber contains collagen with good biocompatibility and biodegradable high molecular weight polymer poly L-lactide-caprolactone (PLCL), which can give the collagen membrane excellent mechanical properties. At the same time, research has found that the ratio of the two plays a key role in the stability of the overall structure of the collagen membrane. When the amount of poly L-lactide-caprolactone added is too large, the fiber diameter of the collagen membrane changes greatly before and after soaking in PBS buffer solution, resulting in poor overall structural stability of the collagen membrane; when the amount of poly L-lactide-caprolactone added is too small, the fiber morphology cannot be observed after the collagen membrane is soaked in PBS buffer solution, which is not conducive to inducing orderly tissue remodeling.

[0015] The purpose of hot pressing is to reduce the gaps between the fibers in the membrane. Under the influence of temperature and pressure, different fibers are connected to a certain extent at the contact points, changing the loose fiber stacking state of the original fiber membrane, thereby improving the mechanical properties of the collagen membrane. The hot pressing temperature is based on the principle of maintaining the integrity of the collagen. The pressure of hot pressing has a significant impact on the thickness of the collagen membrane and can be selected according to the actual requirements of different collagen membranes.

[0016] It should also be noted that the above-mentioned hot pressing treatment also includes vacuum drying and sterilization treatment; specifically, the vacuum pressure of the vacuum drying is 5 to 15 kPa, and the number of cycles is 3 to 7 times; the sterilization treatment can be radiation sterilization.

[0017] The above-mentioned collagen can be derived from one or more of bovine tendon, porcine dermis, fish skin tissue and small intestine; the LA:CL in the poly L-lactide-caprolactone (PLCL) is (15-30): (70-85) (molar ratio); optionally, the molecular weight Mw of the poly L-lactide-caprolactone is 140,000-180,000, for example, the molecular weight Mw is 161,000.

[0018] Preferably, the mass ratio of the collagen to poly (L-lactide-caprolactone) is 1:(1.8-2.2). More preferably, the mass ratio of the collagen to poly (L-lactide-caprolactone) is 1:2.

[0019] Specifically, the average thickness of the fiber membrane is 50 to 300 μm. The thickness of the fiber membrane will affect the thickness of the collagen membrane after the hot pressing treatment, thereby affecting its repair effect.

[0020] Optionally, the average thickness of the fiber membrane is 135-289 μm, 147 μm, 176 μm, 182 μm or 203 μm.

[0021] In a specific embodiment, the temperature of the hot pressing treatment is 40-48°C, the time is 15-40 minutes, and the pressure of the hot pressing treatment is 100-160 Pa. Alternatively, the temperature is 40-44°C, the time is 20-30 minutes; the temperature is 40°C, the time is 30 minutes; the temperature is 42°C, the time is 25 minutes; the temperature is 44°C, the time is 20 minutes.

[0022] Specifically, the hot pressing process involves applying pressure to the fiber membrane using frosted glass at 40-48°C and 100-160 Pa for 15-40 minutes. The hot pressing temperature significantly impacts the performance of the collagen membrane, altering the molecular chain dynamics within poly (L-lactide-caprolactone) and the state of the collagen (whether denaturation occurs). A hot pressing temperature of 40-48°C effectively ensures the hot pressing effect, resulting in the collagen membrane possessing superior mechanical properties.

[0023] In a specific embodiment, any fiber membrane that meets the above conditions can be used in the present invention. For example, the fiber membrane can be prepared by electrospinning.

[0024] Specifically, the spinning solution for electrospinning consists of collagen, poly (L-lactide-caprolactone), hexafluoroisopropanol, acetic acid and water. Collagen with good biocompatibility and biodegradable polymer poly (L-lactide-caprolactone) (PLCL) are the main components, acetic acid and highly polar solvent hexafluoroisopropanol are the auxiliary components, and an appropriate amount of water is added to form a spinning solution that is transparent, evenly dispersed and free of impurities visible to the naked eye.

[0025] Specifically, the mass concentration of collagen in the spinning solution is 5-10 g / mL, the mass concentration of poly (L-lactide-caprolactone) is 10-20 g / mL, the volume concentration of hexafluoroisopropanol is 90%-98%, and the volume concentration of acetic acid is 1%-5%.

[0026] The specific operation of electrospinning is as follows:

[0027] The spinning solution is added to a syringe and connected to a spinneret. Using a drum as a receiving device and a propulsion pump as a propulsion device, high-pressure spinning is applied to the drum to produce a fiber membrane. The surface of the fiber membrane is smooth and free of visible impurities. Optionally, the electrospinning voltage is 25-40 kV, the propulsion rate is 10-30 μL / min, and the distance between the spinneret and the drum is 15-30 cm. By controlling the total amount of spinning solution, the spinning time and, therefore, the thickness of the fiber membrane can be controlled.

[0028] The present invention also protects a collagen membrane prepared by the above preparation method and having the function of inducing orderly tissue remodeling.

[0029] The use of the above-mentioned collagen membrane with the function of inducing orderly tissue remodeling in the preparation of tissue regeneration membranes is also within the scope of protection of the present invention.

[0030] The present invention has the following beneficial effects:

[0031] The collagen membrane of the present invention is made of collagen with good biocompatibility and tissue repair promoting effect, and a high molecular polymer with biodegradability, excellent mechanical properties and good cell adhesion. The combination of poly (L-lactide) and caprolactone not only has an extracellular matrix-like structure, but also has good structural stability, which is beneficial to regulating cell behavior and tissue regeneration. It can effectively promote the growth, adhesion and proliferation of host cells, thereby inducing orderly remodeling of tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG1 is a scanning electron microscope (SEM) image of the collagen membrane of the present invention at different magnifications;

[0033] FIG2 is a scanning electron microscope (SEM) image of the collagen membrane before and after soaking in PBS buffer solution in Example 3 and Comparative Examples 1-2;

[0034] FIG3 is a graph showing changes in fiber diameter before and after the collagen membrane is soaked in PBS buffer solution in Example 3 and Comparative Examples 1-2;

[0035] FIG4 is a graph showing the in vitro cytotoxicity experiments of the collagen membranes in Example 3 and Comparative Examples 1-2. DETAILED DESCRIPTION

[0036] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0037] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0038] The collagen is derived from bovine tendon and is manufactured by Guangzhou Chuanger Biotechnology Co., Ltd. It is called type I collagen powder.

[0039] Poly (L-lactide-caprolactone) 1, molecular weight Mw = 161,000, LA:CL = 70:30, manufactured by Evonik Specialty Chemicals (Shanghai) Co., Ltd., brand LC703S;

[0040] Poly (L-lactide-caprolactone 2), LA:CL=85:15, manufactured by Corbion-Purac, the Netherlands, with the brand name PLC 8516.

[0041] The fiber membrane of the present invention can be prepared by electrospinning:

[0042] The spinning solution (40 mL) was added to the syringe and connected to the spinneret. The roller was used as the receiving device and the propulsion pump was used as the propulsion device to prepare the fiber membrane. The specific parameters of electrospinning were: voltage of 25-40 kV, propulsion rate of 10-30 μL / min, and the distance between the spinneret and the roller was 15-30 cm.

[0043] The spinning solution is composed of collagen, poly L-lactide-caprolactone, hexafluoroisopropanol, acetic acid and water, among which hexafluoroisopropanol, acetic acid and water are solvents.

[0044] Collagen mass concentration (g / mL) = collagen mass / solvent volume*100%;

[0045] PLCL mass concentration (g / mL) = PLCL mass / solvent volume * 100%;

[0046] The concentration of each component is shown in Table 1:

[0047] Table 1

[0048] Example 1

[0049] A method for preparing a collagen membrane capable of inducing orderly tissue remodeling comprises the following steps:

[0050] The fiber membrane 1 is subjected to hot pressing for 30 minutes at 40° C. and 100-160 Pa, vacuum drying, and sterilization to obtain a collagen membrane capable of inducing orderly tissue remodeling.

[0051] Example 2

[0052] A method for preparing a collagen membrane capable of inducing orderly tissue remodeling comprises the following steps:

[0053] The fiber membrane 2 is subjected to hot pressing for 25 minutes at 42° C. and 100-160 Pa, and then vacuum dried and sterilized to obtain a collagen membrane capable of inducing orderly tissue remodeling.

[0054] Example 3

[0055] A method for preparing a collagen membrane capable of inducing orderly tissue remodeling comprises the following steps:

[0056] The fiber membrane 3 is subjected to hot pressing for 20 minutes at 44° C. and 100-160 Pa, vacuum dried, and sterilized to obtain a collagen membrane capable of inducing orderly tissue remodeling.

[0057] Example 4

[0058] A method for preparing a collagen membrane capable of inducing orderly tissue remodeling comprises the following steps:

[0059] The fiber membrane 4 is subjected to hot pressing for 20 minutes at 44° C. and 100-160 Pa, and then vacuum dried and sterilized to obtain a collagen membrane capable of inducing orderly tissue remodeling.

[0060] Example 5

[0061] A method for preparing a collagen membrane capable of inducing orderly tissue remodeling comprises the following steps:

[0062] The fiber membrane 5 is subjected to hot pressing treatment at 44° C. and 100-160 Pa for 20 minutes, vacuum dried, and sterilized to obtain a collagen membrane capable of inducing orderly tissue remodeling.

[0063] Example 6

[0064] A method for preparing a collagen membrane capable of inducing orderly tissue remodeling comprises the following steps:

[0065] The fiber membrane 6 is subjected to hot pressing for 20 minutes at 44° C. and 100-160 Pa, and then vacuum dried and sterilized to obtain a collagen membrane capable of inducing orderly tissue remodeling.

[0066] Comparative Example 1

[0067] A method for preparing a collagen membrane capable of inducing orderly tissue remodeling comprises the following steps:

[0068] The fiber membrane 7 is subjected to hot pressing for 20 minutes at 44° C. and 100-160 Pa, vacuum dried, and sterilized to obtain a collagen membrane capable of inducing orderly tissue remodeling.

[0069] Comparative Example 2

[0070] A method for preparing a collagen membrane capable of inducing orderly tissue remodeling comprises the following steps:

[0071] The fiber membrane 8 is subjected to hot pressing treatment at 44° C. and 100-160 Pa for 20 minutes, vacuum dried, and sterilized to obtain a collagen membrane capable of inducing orderly tissue remodeling.

[0072] Performance Testing

[0073] (1) Collagen membrane SEM test: With reference to ISO 10993-19 “Biological evaluation of medical devices — Part 19: Characterization of physical, chemical, morphological and surface properties of materials”, the fiber morphology of the collagen membrane was observed using a cold field emission scanning electron microscope.

[0074] The collagen membranes of Examples 1-6 and Comparative Examples 1-2 were immersed in a 37°C PBS buffer solution for 24 hours, then removed and dried. The fiber morphology of each sample before and after immersion was observed using a scanning electron microscope (SEM). Ten randomly selected fibers were measured for fiber diameter using a NanoMeasure scanner. The average fiber diameter was calculated as: (average fiber diameter after immersion - average fiber diameter before immersion) / average fiber diameter before immersion * 100%. Since the fibers swell and shrink during immersion, the change rate is considered positive. The test results are shown in Table 2.

[0075] Table 2

[0076] As shown in FIG1 , the collagen membrane of the present invention has an extracellular matrix (ECM)-like microstructure, which can provide a favorable regenerative environment for the growth, adhesion and proliferation of host cells, thereby inducing orderly remodeling of tissues.

[0077] As shown in Table 2 and Figure 3, the average fiber diameter changes in Examples 1-6 after soaking in PBS buffer solution ranged from 6.5% to 10.9%, demonstrating excellent structural stability. In contrast, the collagen membrane of Comparative Example 1, in which the fibers were directly dissolved in PBS buffer solution, exhibited a significant change in average fiber diameter of 59.4% after soaking. This indicates that the collagen membranes of Comparative Example 2 exhibited poor structural stability.

[0078] At the same time, as shown in Figure 2, after the collagen membranes in Example 3, Comparative Example 1 and Comparative Example 2 were soaked in PBS buffer solution, when the mass ratio of collagen to PLCL was 1:1, the fibers of the collagen membrane dissolved and the fiber morphology could not be observed; when the mass ratio of collagen to PLCL was 1:3, the fiber diameter of the collagen membrane changed significantly, that is, the performance was unstable; and when the mass ratio of collagen to PLCL was 1:2, the fiber diameter distribution and size of the collagen membrane before and after soaking in PBS buffer solution did not change substantially, indicating that its performance was stable. Therefore, only when the mass ratio of collagen to PLCL is in an appropriate range can the structural performance of the collagen fiber membrane be stable.

[0079] (2) Cytocompatibility test: Referring to ISO 10993-5 "Biological evaluation of medical devices - Part 5: In vitro cytotoxicity test", the CCK-8 and MTT methods were used to observe the effects of the collagen membrane extracts in Example 3, Comparative Example 1, and Comparative Example 2 on the proliferation rate of mouse epithelial-like fibroblasts to test the cytocompatibility of the collagen membrane.

[0080] The collagen membranes from Example 3, Comparative Example 1, and Comparative Example 2 were completely immersed in MEM medium for 72 hours. The extracts were adjusted to four different concentrations (100%, 75%, 50%, and 25%) for testing. Mouse epithelial fibroblasts were cultured for 24 hours and mixed with the test extracts at different concentrations. The absorbance wavelengths were measured using the CKK-8 and MTT assays, respectively.

[0081] As shown in Figure 4, this experiment studied the survival of L-929 cells in collagen membrane extracts. Compared with the normal control group, the cell survival rates of the negative control group and the positive control group were within the normal range, proving the effectiveness of the experimental results. With the change of the concentration of the collagen membrane extract, the cell proliferation rate was slightly different, but all were greater than 70%. The results showed that the collagen membranes formed in each proportion were not cytotoxic, preliminarily proving their biocompatibility and safety. The collagen membrane prepared by the present invention with the function of inducing orderly tissue remodeling has good structural stability and cell compatibility, which is beneficial to the growth, adhesion and proliferation of host cells, thereby inducing orderly tissue remodeling.

[0082] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for preparing a collagen membrane having the effect of inducing orderly tissue remodeling, characterized in that: The following steps are involved: The fiber membrane is subjected to heat pressing to obtain a collagen membrane that can induce orderly tissue remodeling; The fiber membrane is formed by cross-layering of fibers with an average diameter of 0.5 to 15 μm; The fiber comprises collagen and poly-L-lactide-caprolactone, and the mass ratio of the collagen to the poly-L-lactide-caprolactone is 1:(1.5-2.5).

2. The preparation method according to claim 1, characterized in that: The mass ratio of the collagen to poly-L-lactide-caprolactone is 1:(1.8-2.2).

3. The preparation method according to claim 1, characterized in that: The thickness of the fiber membrane is 50-300 μm.

4. The preparation method according to claim 1, characterized in that: The temperature of the hot pressing treatment is 40-48° C., and the time is 15-40 minutes.

5. The preparation method according to claim 4, characterized in that: The pressure of the hot pressing treatment is 100-160Pa.

6. The preparation method according to claim 1, characterized in that: The fiber membrane is prepared by electrostatic spinning, and the spinning solution of the electrostatic spinning consists of collagen, poly-L-lactide-caprolactone, hexafluoroisopropanol, acetic acid and water.

7. The preparation method according to claim 6, characterized in that: The mass concentration of the collagen is 5-10 g / mL, and the mass concentration of poly-L-lactide-caprolactone is 10-20 g / mL.

8. The preparation method according to claim 6 or 7, characterized in that: The voltage of the electrostatic spinning is 25-40 kV.

9. A collagen membrane having the function of inducing orderly tissue remodeling obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the collagen membrane having the function of inducing orderly tissue remodeling according to claim 9 in preparing a tissue regeneration membrane.

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