Silk fibroin soft tissue patch and preparation method and application thereof

Abstract

The application aims to achieve the anti-adhesion effect through double heterogeneity while ensuring the overall performance of the patch, and specifically provides a silk fibroin soft tissue patch as well as a preparation method and application thereof, the preparation method comprising the following steps: S1, degumming and cleaning silk mesh to obtain a silk fibroin mesh; S2, heat setting the silk fibroin mesh to obtain a set mesh; S3, impregnating the set mesh with a silk fibroin solution and drying to obtain an impregnated mesh; and S4, performing a regulation treatment on the impregnated mesh to obtain a soft tissue patch, wherein the regulation treatment in step S4 is a water vapor crystallization regulation treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, and relates to a silk fibroin soft tissue patch, its preparation method and application. Background Technology

[0002] Soft tissue defects and weaknesses in areas such as the abdominal wall, esophageal hiatus, diaphragm, pelvic floor, tendons, and ligaments are common surgical conditions. Currently, soft tissue repair patches are often used to compensate for and strengthen damaged tissues; therefore, an ideal patch needs to maintain sufficient strength. With the increasing prevalence of minimally invasive surgery, patches also need a certain degree of rigidity to facilitate laparoscopic manipulation. Simultaneously, excellent softness, appropriate toughness, and biodegradability are required to provide postoperative comfort and reduce the risk of long-term foreign body sensation and chronic pain. However, existing patch materials often fail to simultaneously meet all these performance requirements.

[0003] Currently, soft tissue repair patches on the market mainly adopt two preparation routes: First, polymeric synthetic patches represented by polypropylene and polyester, although with excellent mechanical properties, their permanent rigid residues act as "foreign bodies" that continuously irritate the host tissue, which is the root cause of long-term complications such as chronic pain, tissue cutting, local stiffness, organ adhesion and erosion; Second, absorbable biological patches represented by animal-derived decellularized matrix, although degradable, have the risk of xenogeneic immunogenicity (such as causing fever and seroma), and the degradation rate is difficult to control, which can easily lead to insufficient strength maintenance and repair failure. They are not suitable for repair scenarios with high mechanical requirements (such as abdominal wall and tendon repair).

[0004] Furthermore, soft tissue patches may cause adhesions during actual use (such as intra-abdominal or tendon applications), thus necessitating anti-adhesion measures. In existing technologies, anti-adhesion repair patches often employ composite structures, adding an additional anti-adhesion layer to the original sheet structure to achieve the anti-adhesion effect. For example, CN115569242A discloses an anti-adhesion abdominal wall hernia composite patch and its preparation method, which achieves the anti-adhesion effect by coating an adhesive onto the smooth surface of the dermal matrix and attaching a biofilm; CN110639065A discloses a bacterial cellulose-based asymmetric bilateral heterogeneous biological patch and its preparation method, which achieves bilateral heterogeneity through the surface of the bacterial cellulose membrane, with one side being an anti-adhesion surface and the other a microporous repair-promoting surface. The anti-adhesion surface is obtained by hydrophobically modifying the bacterial cellulose surface with a long-chain hydrophobic modifier.

[0005] While composite meshes can achieve physical isolation, the interface between heterogeneous materials carries the risk of delamination under dynamic stress within the body, and their poor flexibility also poses challenges for minimally invasive surgery. The microporous structure of bacterial cellulose meshes is not conducive to deep integration between the mesh and tissue, and the residual hydrophobic modifiers also pose a potential toxicity risk. Summary of the Invention

[0006] To address the aforementioned issues and ensure the overall performance of the patch while achieving anti-adhesion through double-sided heterogeneity, this invention provides a method for preparing a silk fibroin soft tissue patch, comprising: step S1, degumming and cleaning a silk mesh to obtain a silk fibroin mesh; step S2, heat-setting the silk fibroin mesh to obtain a shaped mesh; step S3, impregnating the shaped mesh with a silk fibroin solution and drying it to obtain an impregnated mesh; and step S4, subjecting the impregnated mesh to a conditioning treatment to obtain a soft tissue patch, wherein the conditioning treatment in step S4 is a water vapor crystallization conditioning treatment.

[0007] Preferably, in the preparation method of silk fibroin soft tissue patch provided by the present invention, the water vapor crystallization control treatment in step S4 is to treat the impregnated mesh at a temperature of 55℃-75℃ and a humidity of 70%-90% for 30min-120min.

[0008] Preferably, in the preparation method of the silk fibroin soft tissue patch provided by the present invention, in step S3, the concentration of the silk fibroin solution is 1%-10%, and the amount of silk fibroin solution absorbed by the shaping mesh is 200%-500% of the dry weight of the mesh.

[0009] Preferably, in the preparation method of the silk fibroin soft tissue patch provided by the present invention, the heat setting process in step S2 is as follows: the edge of the silk fibroin mesh is fixed on a hollow square fixing clamp, so that the mesh is stretched straight in the warp and weft directions. At this time, the tightness of the fixation is such that the center of the stretched mesh can withstand a pressure of 2N-5N without collapsing. After fixing, it is set at a temperature of 45℃-65℃ for 30min-3h.

[0010] This invention also provides a silk fibroin soft tissue patch, prepared using the preparation method described in any of the preceding claims. Furthermore, this invention provides the application of this silk fibroin soft tissue patch in the preparation of soft tissue repair and reinforcement materials, wherein the soft tissue repair and reinforcement materials are used for soft tissue reinforcement and repair in the treatment of chest and abdominal wall defects, abdominal wall hernias, hiatal hernias, diaphragmatic hernias, pelvic floor hernias, inguinal hernias, tendon and ligament injuries, etc.

[0011] According to the preparation method of the silk fibroin soft tissue patch provided by the present invention, after the silk fibroin mesh is shaped, it is impregnated with a silk fibroin solution and further subjected to water vapor crystallization regulation treatment to form a bi-sided heterogeneous structure with one side rough and the other side smooth. This achieves the dual function of preventing adhesion on the smooth side and promoting tissue regeneration on the rough side. In addition, the water vapor crystallization regulation treatment is mild and controllable, and the overall performance parameters of the patch, such as softness, stiffness, resistance to plastic deformation, and resilience, are improved to a certain extent. While achieving the aforementioned dual function, it can ensure the surgical operability and repair performance of the patch itself. It is suitable for the repair of soft tissues such as chest and abdominal wall defects, abdominal wall hernias, hiatal hernias, diaphragmatic hernias, pelvic floor hernias, inguinal hernias, and tendon and ligament injuries, and has broad clinical application prospects and market potential. Attached Figure Description

[0012] Figure 1 This is a graph showing the surface roughness test results of Test Example 1 of the present invention.

[0013] Figure 2 This is a surface SEM image of the soft tissue patch of Embodiment 2 of the present invention.

[0014] Figure 3 yes Figure 2 Local SEM images of the soft tissue patch in Example 2.

[0015] Figure 4 yes Figure 3 A magnified view of the area within the dashed box.

[0016] Figure 5 This is a graph showing the test results of the frontal dynamic friction coefficient of Test Example 3 of the present invention.

[0017] Figure 6 This is a photograph of the simulated unfolding of the soft tissue patch in Test Example 4 of this invention.

[0018] Figure 7 This is a photograph of the implantation test of Test Example 4 of the present invention.

[0019] Figure 8 This is a graph showing the softness test results of Test Example 7 of the present invention.

[0020] Figure 9 This is a graph showing the stiffness test results of Test Example 8 of the present invention.

[0021] Figure 10 This is a graph showing the results of the plastic deformation test of Test Example 9 of the present invention.

[0022] Figure 11 This is a graph showing the test results of deformation hysteresis in Test Example 10 of the present invention.

[0023] Figure 12This is a diagram showing the postoperative observation results of abdominal wall defect repair in Test Example 11 of this invention.

[0024] Figure 13 This is a histological result image of abdominal wall defect repair after test example 11 of the present invention. Detailed Implementation

[0025] The specific embodiments of the present invention are described below with reference to the accompanying drawings and examples. In each embodiment, the raw materials without specified sources are all commercially available, and the experimental operations not described in detail are all performed in accordance with conventional practices in the art. The silk mesh in each embodiment is obtained by warp knitting of mulberry silk.

[0026] Example 1

[0027] This embodiment provides a method for preparing a silk fibroin soft tissue patch (hereinafter referred to as a soft tissue patch), including the following steps:

[0028] S1. Degumming and washing the silk mesh to obtain silk fibroin mesh. The specific operation is as follows: immerse in Na2CO3 solution and heat for 20 min, repeat 3 times, and then wash 3 times with purified water. The Na2CO3 concentration is 0.2%, and the degumming temperature is 87℃.

[0029] S2. The silk fibroin mesh obtained in step S1 is heat-set to obtain a shaped mesh. Specifically, the edges of the silk fibroin mesh are fixed to a hollow square clamp, ensuring the mesh is taut in both warp and weft directions and that its center can withstand a 5 N pressure without collapsing. Heat-set at 60℃ for 60 minutes. The specific control method is as follows: a 5 N weight is placed at the center of the silk fibroin mesh, and the degree of collapse is observed. The tightness of the fixation is adjusted until the requirement is met (i.e., no collapse).

[0030] S3. The shaped mesh obtained in step S2 is impregnated with silk fibroin solution and dried to obtain the impregnated mesh. The specific operation is as follows: Prepare a 1% silk fibroin solution, weigh the shaped mesh, and weigh 275% of the silk fibroin solution according to the mass of the shaped mesh. Immerse the shaped mesh in the silk fibroin solution until the shaped mesh has completely absorbed the solution. Then, lay the impregnated mesh flat on a smooth glass plate and dry for 30 minutes.

[0031] S4. The impregnated mesh obtained in step S3 is subjected to conditioning treatment to obtain a soft tissue patch. This conditioning treatment is a water vapor crystallization conditioning treatment. The specific operation is as follows: the impregnated and dried mesh and the glass plate are placed together in a constant temperature and humidity chamber set at 55℃ and 85% humidity, and left to stand for 40 minutes. The surface in contact with the glass plate (the downward-facing surface) is the front side of the soft tissue patch, and the upward-facing surface is the back side of the soft tissue patch.

[0032] Example 2

[0033] This embodiment provides another method for preparing soft tissue patches. In this embodiment, steps S1-S2 are the same as in Example 1, but steps S3-S4 are different from those in Example 1, as detailed below:

[0034] S3. The shaped mesh obtained in step S2 is impregnated with a silk fibroin solution and dried to obtain an impregnated mesh. The specific operation is as follows: Prepare a 5% silk fibroin solution, weigh the shaped mesh, and weigh 350% of the silk fibroin solution according to the mass of the shaped mesh. Immerse the shaped mesh in the silk fibroin solution until the mesh completely absorbs the solution. Then, lay the impregnated mesh flat on a smooth glass plate and dry for 45 minutes.

[0035] S4. The impregnated mesh obtained in step S3 is processed to obtain a soft tissue patch. The specific operation is as follows: the impregnated and dried mesh and the glass plate are placed in a constant temperature and humidity chamber with a temperature of 65°C and a humidity of 85% and left to stand for 90 minutes.

[0036] Example 3

[0037] This embodiment provides yet another method for preparing a soft tissue patch. In this embodiment, steps S1-S2 are the same as in Example 1, but steps S3-S4 are different from those in Example 1, as detailed below:

[0038] S3. The shaped mesh obtained in step S2 is impregnated with a silk fibroin solution and dried to obtain an impregnated mesh. The specific operation is as follows: Prepare an 8% silk fibroin solution, weigh the shaped mesh, and weigh 400% of the silk fibroin solution according to the mass of the shaped mesh. Immerse the shaped mesh in the silk fibroin solution so that the shaped mesh completely absorbs the solution. Then, lay the impregnated mesh flat on a smooth glass plate and dry for 50 minutes.

[0039] S4. The impregnated mesh obtained in step S3 is processed to obtain a soft tissue patch. The specific operation is as follows: the impregnated and dried mesh and the glass plate are placed together in a constant temperature and humidity chamber with a temperature of 75°C and a humidity of 90% and left to stand for 120 minutes.

[0040] Comparative Example 1

[0041] In the soft tissue patch preparation method of this comparative example, steps S1-S3 are the same as in Example 1, but the subsequent step S4 is not performed. That is, the soft tissue patch of this comparative example is essentially the shaped mesh in Example 1, which has not undergone impregnation, drying, or conditioning treatment.

[0042] Comparative Example 2

[0043] In the soft tissue patch preparation method of this comparative example, steps S1-S2 are the same as in Example 1, but step S4 is performed directly after step S2. That is, in this comparative example, the shaped mesh obtained in step S2 is not subjected to impregnation and drying treatment, and is directly subjected to the conditioning treatment in step S4.

[0044] Comparative Example 3

[0045] In the soft tissue patch preparation method of this comparative example, steps S1-S2 are the same as in Example 1, but steps S3-S4 are different from those in Example 1, as detailed below:

[0046] S3. The shaped mesh obtained in step S2 is impregnated with silk fibroin solution and dried to obtain the impregnated mesh. The specific operation is as follows: prepare a 1% silk fibroin solution, weigh the shaped mesh, weigh the silk fibroin solution according to 275% of the mesh mass, and immerse the shaped mesh in the silk fibroin solution so that the shaped mesh completely absorbs the solution.

[0047] S4. Dry the silk fibroin mesh obtained in step S3 at 40°C for 24 hours to obtain the final product.

[0048] Comparative Example 4

[0049] This comparative example is a commercially available composite hernia repair patch that can be used intra-abdominally. The product has a two-layer structure, including a polypropylene monofilament woven mesh and an absorbable membrane made of poly(propylene-caprolactone) (hereinafter referred to as the polypropylene patch).

[0050] Comparative Example 5

[0051] In the soft tissue patch preparation method of this comparative example, steps S1-S2 are the same as in Example 1, but steps S3-S4 are different from those in Example 1, as detailed below:

[0052] S3. The shaped mesh obtained in step S2 is impregnated with a silk fibroin solution and dried to obtain an impregnated mesh. The specific operation is as follows: Prepare a 0.5% silk fibroin solution, weigh out the shaped mesh, and weigh out 275% of the silk fibroin solution according to the mass of the shaped mesh. Immerse the shaped mesh in the silk fibroin solution until the shaped mesh completely absorbs the solution. Then, lay the impregnated mesh flat on a smooth glass plate and dry for 30 minutes.

[0053] S4. The impregnated mesh obtained in step S3 is processed to obtain a soft tissue patch. The specific operation is as follows: the impregnated mesh is placed in a constant temperature and humidity chamber with a temperature of 55℃ and a humidity of 90% and left to stand for 120 min.

[0054] Comparative Example 6

[0055] In the soft tissue patch preparation method of this comparative example, steps S1-S2 are the same as in Example 1, but steps S3-S4 are different from those in Example 1, as detailed below:

[0056] S3. The shaped mesh obtained in step S2 is impregnated with silk fibroin solution and dried to obtain an impregnated mesh. The specific operation is as follows: Prepare a 12% silk fibroin solution, weigh the shaped mesh, and weigh 400% of the silk fibroin solution according to the mass of the shaped mesh. Immerse the shaped mesh in the silk fibroin solution until the shaped mesh completely absorbs the solution. Then, lay the impregnated mesh flat on a smooth glass plate and dry for 45 minutes.

[0057] S4. The impregnated mesh obtained in step S3 is processed to obtain a soft tissue patch. The specific operation is as follows: the impregnated mesh is placed in a constant temperature and humidity chamber with a temperature of 60℃ and a humidity of 90% and left to stand for 120 min.

[0058] Test Example 1

[0059] This test example is a surface roughness test of a soft tissue patch.

[0060] In this test example, the roughness of the front and back surfaces of each embodiment and comparative example was tested using a KES-FB4 surface roughness tester. The results are shown in Table 1 below:

[0061] Table 1 Surface roughness of soft tissue patches

[0062]

[0063] Figure 1 This is a graph showing the surface roughness test results of Test Example 1 of the present invention.

[0064] As shown in Table 1 above and Figure 1 As shown, compared to Comparative Example 3, the roughness of the front and back surfaces of each soft tissue patch in Examples 1-3 are significantly different.

[0065] Test Example 2

[0066] This test case is a surface electron microscopy examination of a soft tissue patch from Example 2.

[0067] Figure 2 This is a surface SEM image of the soft tissue patch of Embodiment 2 of the present invention. Figure 2 In the image, the left side shows the front of the soft tissue patch, and the right side shows the back of the soft tissue patch.

[0068] like Figure 2 As shown, the front and back of the soft tissue patch have different weaving structures, which is caused by the weaving method of the silk mesh.

[0069] Figure 3 yes Figure 2 Magnified SEM image of the soft tissue patch in Example 2. Figure 4 yes Figure 3 A magnified view of the area within the dashed box. Figure 3 In the middle, the left side shows the front of the soft tissue patch, and the right side shows the back of the soft tissue patch; Figure 4 In the middle, on the left is Figure 3 The enlarged view within the dashed box on the left, and the view on the right. Figure 3 A magnified view of the area within the dashed box on the right.

[0070] like Figures 2-4 As shown, the fibers in the mesh are intertwined and bonded together, forming a coarser, near-fibrous structure. This state is caused by the adsorbed silk fibroin solution being treated with water vapor crystallization and then dried.

[0071] Combining the surface roughness test results of Comparative Example 1 in Table 1 of Test Example 1, it can be seen that when only simple shaping treatment is performed, the roughness of the front and back sides of the mesh is relatively similar, indicating that the difference in the weaving structure of the front and back sides has little impact on the roughness of the silk mesh. However, after impregnation with silk fibroin solution and water vapor crystallization regulation treatment, a large difference in roughness between the front and back sides occurs. This may be because water vapor crystallization regulation treatment can promote the transformation of the α-helix in the adsorbed silk fibroin structure to β-sheet, increasing its crystallinity. In the mesh structure, the interaction between the silk fibers and silk fibroin on the two surfaces is different, thus forming bifacial heterogeneity, resulting in a large difference in roughness between the front and back sides.

[0072] In practical applications, the rough surface can promote the adhesion of rough surface cells, tissue ingrowth and integration, and can induce fibroblasts and other cells to enter the patch to form a strong "tissue-patch" complex, achieving strong biological fixation and effectively reducing the risk of patch displacement and recurrence; the smooth surface can inhibit tissue adhesion, effectively preventing the formation of pathological adhesions between internal organs and the patch, and avoiding serious postoperative complications such as intestinal obstruction and chronic pain.

[0073] Test Example 3

[0074] This test case is a test of the coefficient of friction of the front surface of a soft tissue patch.

[0075] This test example specifically uses the KES-FB4 surface tester to test the frontal dynamic friction coefficient of each embodiment and comparative example. This dynamic friction coefficient can reflect the smoothness of the surface, and the results are shown in Table 2 below:

[0076] Table 2. Coefficient of dynamic friction on the front of the patch

[0077]

[0078] Figure 5 This is a graph showing the test results of the front surface friction coefficient of Test Example 3 of the present invention.

[0079] As shown in Table 2 above and Figure 5 As shown in the results of Examples 1-3, the dynamic friction coefficient of the soft tissue patch gradually decreases with increasing concentration of the silk fibroin solution during impregnation. Comparative Example 3 employed a treatment method of further heating and drying after impregnation, which, while improving the crystallinity of the adsorbed silk fibroin to some extent, resulted in an excessively high dynamic friction coefficient on the front of the patch, indicating that the smoothness of the patch's surface was not significantly improved. This phenomenon demonstrates that using water vapor crystallization control can effectively improve the smoothness of the front surface; by facing this side towards the organ during use, postoperative adhesions can be effectively reduced.

[0080] Furthermore, comparing Examples 1-3 with Comparative Example 5, it can be seen that although Comparative Example 5 used the same impregnation treatment and water vapor crystallization control treatment as Examples 1-3, its impregnation solution concentration was lower, resulting in a higher frontal dynamic friction coefficient of the final soft tissue patch. Therefore, the concentration of silk fibroin solution in the impregnation treatment should not be too low and should be set to not less than 1%.

[0081] Test Example 4

[0082] This test case is a surgical operability test of soft tissue patches.

[0083] (1) Simulated deployment

[0084] During laparoscopic surgery, to insert the patch into the body, it is often necessary to roll the patch, secure it with sutures at concentric points, and then deliver it to the surgical area through a trocar. To simulate the unfolding of soft tissue patches in both dry and wet states, the soft tissue patches in each embodiment and comparative example were gently manipulated with surgical instruments. After moistening each soft tissue patch with saline and rolling it up, it was gently manipulated again with surgical instruments to assess the operability of patch unfolding.

[0085] Figure 6 This is a photograph of the simulated unfolding of the soft tissue patch in Test Example 4 of this invention.

[0086] like Figure 6 As shown, in a dry state, all embodiments and comparative examples 3, 4, and 6 can be smoothly unfolded like a roller shutter door; after being moistened, comparative examples 1, 2, and 5 are too soft and have low curling tension, making it difficult to unfold smoothly after being curled, which will be difficult and time-consuming to operate in a confined space.

[0087] (2) Implantation trial

[0088] Laparoscopic patch implantation experiments were conducted on miniature pigs. Specifically, the same operator and the same technique were used to fix the soft tissue patches of Example 2 and Comparative Example 4 onto the abdominal wall of the pigs.

[0089] Figure 7 This is a photograph of the implantation test of Test Example 4 of the present invention.

[0090] like Figure 7 As shown, the soft tissue patches of Example 2, Comparative Example 1, and Comparative Example 4 were all successfully deployed and fixed under laparoscopy. The implantation and fixation time for Example 2 was 8.5 ± 2.3 min, and for Comparative Example 4 it was 8.1 ± 1.9 min, which were similar. In addition, Comparative Example 1 showed edge curling and unevenness after fixation; however, Example 2 did not exhibit this phenomenon, and the patch edges were smooth and without curling after fixation.

[0091] Test Example 5

[0092] Pore ​​size and porosity are key factors affecting tissue regeneration. This test case is a test of the pore size, porosity and weight per unit area of ​​a soft tissue patch.

[0093] This test case uses the following method for testing:

[0094] (1) Aperture

[0095] The aperture was measured using a micrometer, and the average value was calculated from 10 randomly selected apertures on the front side.

[0096] (2) Porosity

[0097] Take a photo of the reverse side of the patch, import it into image processing software to convert it into a black and white image. The black areas in the image represent mesh holes. Calculate the proportion of the black area to the total area of ​​the patch, which is the porosity.

[0098] (3) Mass per unit area

[0099] The product was dried in an oven at 105 °C until constant weight, weighed using an analytical balance, and the mass per unit area was calculated.

[0100] The test results are shown in Table 3 below:

[0101] Table 3 Pore size, porosity, and mass per unit area

[0102]

[0103] As shown in Table 3 above, except for Comparative Example 4, the pore size and porosity of each embodiment are not significantly different from those of the comparative examples, indicating that the conditioning treatment does not affect the pore size and porosity of the patch. Furthermore, the pore size of the soft tissue patches in each embodiment is between 0.2 mm × 0.2 mm and 0.5 mm × 0.5 mm, and the porosity is 10-30%, a range suitable for tissue cell and blood vessel ingrowth.

[0104] Test Example 6

[0105] An ideal patch needs to have sufficient mechanical properties to prevent hernia recurrence. This test case is a test of the mechanical properties of a soft tissue patch.

[0106] This test case uses the following method for testing:

[0107] (1) Tensile strength

[0108] Tensile strength (N / cm) is the maximum force required to stretch a patch to break under specified conditions. The test is performed using an electronic universal testing machine. A sample 100mm long and 20mm wide is prepared, with a 50mm gap between the two clamps. The sample is stretched at a speed of 100mm / min until it breaks, and the tensile strength (N / cm) is recorded.

[0109] (2) Tear strength

[0110] Tear strength refers to the force that a patch can withstand when it is torn. The test is conducted using an electronic universal testing machine. A 100mm × 50mm trouser-shaped tear sample is prepared, with a 50mm long cut made along the center of the width direction. The sample is stretched at a tensile speed of 100mm / min until it is completely torn, and the maximum tear strength (N) is recorded.

[0111] (3) Bursting strength

[0112] Bursting strength is the maximum force measured when a spherical plunger presses against a sample perpendicular to its plane until it breaks. The implanted patch experiences varying stresses from abdominal pressure due to different patient activities (such as coughing, sitting, standing, and walking), and bursting strength affects post-implantation repair outcomes and recurrence rates. The patch is held flat, tension-free, and wrinkle-free on a clamp, a 25mm diameter spherical plunger is selected, and the bursting speed is 300mm / min. The maximum bursting strength (N) is recorded.

[0113] The mechanical property test results are shown in Table 4 below:

[0114] Table 4 Tensile strength, tear strength, bursting strength

[0115]

[0116] As shown in the table above, the conditioning treatment has no effect on the mechanical properties of the patch, and the tensile strength in both warp and weft directions of the patches in each embodiment is greater than 40 N / cm, the tear strength in both warp and weft directions is greater than 10 N / cm, and the burst strength is greater than 300 N. The tensile strength of the prosthesis used for hernia repair should not be lower than the same tensile strength of the human abdominal wall, and the maximum tensile strength of the human abdominal wall ranges from 11 N / cm to 27 N / cm. Therefore, each embodiment can provide sufficient mechanical strength for abdominal wall repair.

[0117] Test Example 7

[0118] Softness reflects how well a mesh adapts to intra-abdominal pressure and compression from surrounding tissues. A mesh with good softness can better accommodate bodily movements, reducing friction and pressure on tissues and nerves, and preventing chronic postoperative pain. This test case demonstrates the softness test of a soft tissue mesh, conducted using the following method:

[0119] The softness of the patch was tested using a TSA softness analyzer in both wet (after implantation and absorption of bodily fluids) and dry states. The wet patch had a water content of 200%-300% (obtained by soaking the dry patch in physiological saline for 5 minutes; the wet patches in the following test examples were prepared using the same method). The test results are shown in Table 5 below. In the table, the lower the softness value, the softer the patch.

[0120] Table 5 Softness

[0121]

[0122] Figure 8 This is a graph showing the softness test results of Test Example 7 of the present invention.

[0123] As shown in Table 5 and Figure 8 As shown, the soft tissue patches of each embodiment already exhibited low softness values ​​in the dry state, which decreased significantly in the wet state, and were significantly lower than those of commercially available polypropylene patches (p < 0.001). This result indicates that the soft tissue patches of the embodiments offer better in-vivo implantation comfort than commercially available polypropylene patches. The wet softness value of Comparative Example 6 was significantly higher than that of Examples 1-3, indicating poor in-vivo implantation comfort and potential for causing a foreign body sensation after implantation. Therefore, the concentration of the silk fibroin solution used for impregnation should not be too high and should be set no higher than 10%.

[0124] In addition, the dry softness values ​​of Examples 1-3 are all higher than those of Comparative Example 3, indicating that the water vapor crystallization control treatment has a certain impact on the softness of the patch; however, the wet softness values ​​of each example are all lower and the difference from Comparative Example 3 is small, indicating that the water vapor crystallization control treatment does not lead to unqualified softness and the resulting soft tissue patch still has very good in vivo implantation comfort.

[0125] Test Example 8

[0126] Rigidity affects the handling performance of patches. Patches with high rigidity resist the compression of the tube better when passing through the trocar, are pushed more smoothly, and are less likely to get stuck; after entering the body, they are more likely to unfold on their own due to their rigidity; and during fixation, they are less likely to curl or wrinkle.

[0127] This test example demonstrates the stiffness (mm / N) of soft tissue patches. Specifically, a TSA softness analyzer was used to test the stiffness of each patch in both dry and wet states. The test results are shown in Table 6 below. In the table, the smaller the stiffness value, the stiffer the patch.

[0128] Table 6 Stiffness

[0129]

[0130] Figure 9 This is a graph showing the stiffness test results of Test Example 8 of the present invention.

[0131] As shown in Table 6 and Figure 9 As shown, the dry and wet stiffness of Examples 1-3 were significantly better than those of Comparative Examples 1-2 (p < 0.001), indicating that water vapor crystallization regulation treatment can effectively improve the stiffness of the patch and effectively optimize surgical performance. Furthermore, the stiffness of Example 3 was comparable to that of Comparative Example 4 (p > 0.05), indicating that the soft tissue patch prepared by the method of the present invention can match the operational performance of commercially available patches.

[0132] Furthermore, the stiffness of Comparative Example 2 under both dry and wet conditions showed no significant difference compared to Comparative Example 1, indicating that directly subjecting the shaped mesh (i.e., the mesh composed of silk fibroin fibers) to steam crystallization treatment without impregnation with silk fibroin solution does not increase stiffness. A comprehensive comparison of Comparative Examples 5-6 with Examples 1-3 also shows that the concentration of the silk fibroin solution during impregnation is generally positively correlated with stiffness in both dry and wet conditions, further demonstrating that silk fibroin solution impregnation is also key to improving stiffness. The stiffness of Comparative Example 5 was not significantly improved compared to Comparative Example 1, indicating that the operability of Comparative Example 5 was not significantly improved. Therefore, the concentration of the silk fibroin solution in the impregnation and crystallization treatment should not be too low, and should be set at no less than 1%. In other words, silk fibroin solution impregnation combined with steam crystallization control treatment can truly improve stiffness.

[0133] Test Example 9

[0134] This test case is a plastic deformation test of a soft tissue patch, as detailed below:

[0135] The stability of the patch's shape is characterized by plastic deformation (μm). The larger the absolute value of the plastic deformation displacement, the worse the patch's deformation recovery ability, which means that it will be gradually stretched, thinned, or even contracted after implantation, leading to changes in the tension of the repair area and increasing the risk of hernia recurrence.

[0136] In this test case, the absolute values ​​of plastic deformation of each patch in both dry and wet states were measured using a TSA softness analyzer. The results are shown in Table 7 below. The larger the absolute value of plastic deformation, the worse the stability of the patch shape.

[0137] Table 7 Absolute values ​​of plastic deformation

[0138]

[0139] Figure 10 This is a graph showing the results of the plastic deformation test of Test Example 9 of the present invention.

[0140] As shown in Table 7 above and Figure 10 As shown, the soft tissue patches of each embodiment exhibited low plastic deformation under both wet and dry conditions, and were significantly lower than those of Comparative Example 1 (p < 0.001). This indicates that water vapor crystallization treatment can effectively improve the stability of patch morphology and prevent patch shrinkage and deformation due to creep relaxation after implantation. The plastic deformation of Comparative Example 3 was higher than that of Example 1 (p < 0.05), indicating that heating and drying crystallization was not effective in improving morphological stability.

[0141] Furthermore, by comparing Comparative Examples 5-6 with Examples 1-3, it can be seen that the concentration of silk fibroin solution during impregnation treatment is correlated with the amount of plastic deformation of the soft tissue patch in both dry and wet states. The higher the concentration of silk fibroin solution, the lower the corresponding amount of plastic deformation in both dry and wet states. This indicates that silk fibroin solution impregnation treatment also has the effect of improving the stability of morphological maintenance. When combined with water vapor crystallization treatment, it can produce a soft tissue patch with excellent morphological maintenance stability and effectively prevent hernia recurrence.

[0142] Test Case 10

[0143] Deformation hysteresis refers to the energy loss of a patch after repeated deformation under external forces (such as breathing, walking, and exercise). A small deformation hysteresis results in low energy loss and good resilience. A patch with good resilience can quickly expand after entering the body, and its energy loss is low under cyclic loads such as breathing and coughing. This reduces tissue fatigue and chronic pain caused by internal friction, and also means that the patch is more sensitive to force and better matches the physiological movements of the abdominal wall.

[0144] This test example uses deformation hysteresis (J) to characterize the resilience of the patch. Both dry and wet patches were tested using a TSA softness analyzer. The results are shown in Table 8 below. In the table, a larger deformation hysteresis value indicates greater deformation hysteresis and poorer resilience of the patch.

[0145] Table 8 Deformation Hysteresis

[0146]

[0147] Figure 11 This is a graph showing the test results of deformation hysteresis in Test Example 10 of the present invention.

[0148] As shown in Table 8 and Figure 11 As shown, in each embodiment, the deformation hysteresis under both dry and wet conditions was less than that of Comparative Example 1 (p < 0.001), and the deformation hysteresis under dry conditions in Examples 2 and 3 was not significantly different from that of commercially available patches (p > 0.05), indicating that crystallization control treatment can improve the resilience of the patches and achieve resilience comparable to that of commercially available patches. Furthermore, the resilience of Example 1 was better than that of Comparative Example 3, indicating that the combination of silk fibroin solution impregnation and water vapor crystallization control treatment can effectively improve the resilience of the patches.

[0149] Test Example 11

[0150] To investigate the safety and effectiveness of soft tissue patches, an abdominal wall defect model was established and hernia repair was performed experimentally.

[0151] The specific operation for establishing and repairing the abdominal wall defect model is as follows: a 3cm × 5cm rectus abdominis muscle defect area is formed on the left and right sides of the abdomen of the same miniature pig. The defect area on the left side is repaired using the patch of Comparative Example 4, and the defect area on the right side is repaired using the soft tissue patch of Example 2.

[0152] Figure 12 This is a diagram showing the postoperative observation results of abdominal wall defect repair in Test Example 11 of this invention.

[0153] like Figure 12 As shown, infection and recurrence were observed in the repair area at 1 month and 6 months postoperatively. No infection or recurrence was observed in either group, and the patches were covered by new tissue.

[0154] Figure 13 This is a histological result image of abdominal wall defect repair after test example 11 of the present invention.

[0155] Animals were sacrificed at 1 month and 6 months post-surgery, and tissue samples from the repaired site were taken for histological evaluation (HE and MASSON staining). The results are as follows: Figure 10As shown: One month post-surgery, both Example 2 and Comparative Example 4 groups had formed neoperitoneum, with the newly formed tissue in the repair area of ​​Example 2 group being denser; Six months post-surgery, the repair area in Example 2 group had been largely replaced by regenerated tissue, with a large number of new blood vessels and new collagen visible, indicating that the patch degradation and tissue remodeling were basically complete; Comparative Example 4 showed no degradation, and the repair area consisted of disordered repair tissue. No adhesion was observed in either group, indicating that Example 2 and commercially available patches have comparable anti-adhesion effects.

[0156] The above results demonstrate that the soft tissue patch of Example 2 exhibits good biocompatibility. In particular, compared to commercially available patches, the soft tissue patch of Example 2 effectively promotes tissue ingrowth, fosters the formation of denser and more orderly new tissue in the repair area, and enhances tissue regeneration.

[0157] Based on the results of the above test cases, the following conclusions can be drawn:

[0158] 1) By impregnating the soft tissue patch with silk fibroin solution and combining it with water vapor crystallization control treatment (various embodiments), a double-sided heterogeneous structure can be given to the soft tissue patch, that is, one side of the patch is smooth, which has the effect of reducing postoperative adhesion, and the other side is rough, which has the effect of promoting tissue ingrowth.

[0159] 2) Compared with the unimpregnated and untreated silk fibroin shaping mesh (i.e., Comparative Example 1), the stiffness of the soft tissue patch can be improved in both dry and wet states by impregnating it with silk fibroin solution and combining it with water vapor crystallization conditioning treatment (each example). In the dry state, it can achieve stiffness comparable to commercially available polypropylene patches, which is beneficial for doctors to cut, hold and place the patch more accurately and familiarly, and the patch is less likely to curl or deform.

[0160] 3) The soft tissue patches of each embodiment exhibit significantly improved softness in the wet state compared to the dry state, and are all superior to the commercially available polypropylene patch of Comparative Example 4. This indicates that these soft tissue patches can switch softness characteristics in a physiological environment, better adapting to the movement and deformation of surrounding tissues, thereby reducing mechanical irritation and foreign body sensation. The absolute values ​​of plastic deformation of the soft tissue patches of each embodiment in both the dry and wet states are lower than those in Comparative Examples 1-3, meaning that the soft tissue patches have significantly enhanced resistance to permanent deformation, laying a solid foundation for long-term stable mechanical support. Similarly, the deformation hysteresis of the soft tissue patches of each embodiment in both the dry and wet states is lower than that in Comparative Examples 1-3, meaning that the patches have a longer fatigue life under dynamic loads and better resistance to deformation.

[0161] 4) Comparative Example 3 involved immersion in a silk fibroin solution followed by heating and drying. This treatment improved the crystallinity of the silk fibroin, but this improvement was unstable. That is, the patch exhibited certain stiffness, resistance to plastic deformation, and resilience in a dry state, but these properties decreased sharply in a wet state. In cases with significant fluid buildup during surgery, the patch's operability was not significantly improved. In contrast, the soft tissue patch of the embodiment retained certain stiffness, resistance to plastic deformation, and resilience in a wet state, which is beneficial for implanting and fixing the patch in a wet environment.

[0162] 5) Under laparoscopy, the operability of the soft tissue patches in each embodiment was no different from that of the patch in Comparative Example 4, and the operation time was similar for both. Furthermore, the repair of the abdominal wall defect model demonstrated that the soft tissue patch of Example 2 has good safety and effectiveness, making it a good product for the repair of abdominal wall and other soft tissues.

[0163] In summary, by impregnating silk fibroin solution and combining it with water vapor crystallization control, the soft tissue patch prepared by this invention achieves an ideal balance of bifacial heterogeneous structure and "rigidity and flexibility." This means it achieves the dual functions of preventing adhesion and promoting tissue regeneration, while simultaneously optimizing softness, stiffness, resistance to plastic deformation, and elastic recovery. This soft tissue patch, which is easy to handle, biocompatible, and effectively promotes tissue regeneration, has broad clinical application prospects and market potential.

Claims

1. A method for preparing a silk fibroin soft tissue patch, characterized in that, include: Step S1: Degumming and washing the silk mesh to obtain silk fibroin mesh; Step S2: Heat-set the silk fibroin mesh to obtain a shaped mesh; Step S3: The shaped mesh is impregnated with silk fibroin solution and dried to obtain an impregnated mesh. The drying includes laying the impregnated mesh flat on a smooth glass plate and drying it. Step S4: The impregnated mesh is subjected to conditioning treatment to obtain a soft tissue patch. In step S4, the conditioning treatment is a water vapor crystallization conditioning treatment. This water vapor crystallization conditioning treatment involves placing the impregnated and dried mesh and glass plate together in a constant temperature and humidity chamber and treating them for 30 min to 120 min at a temperature of 55℃-75℃ and a humidity of 70%-90%. In step S3, the concentration of the silk fibroin solution is 1%-10%. The amount of silk fibroin solution absorbed by the shaping mesh is 200%-500% of the dry weight of the mesh.

2. The method for preparing silk fibroin soft tissue patch according to claim 1, characterized in that: in, The heat setting process in step S2 is as follows: The edges of the silk fibroin mesh are fixed to hollow square clamps, making the silk fibroin mesh taut in both warp and weft directions. The tightness of the fixation is such that the center of the taut silk fibroin mesh can withstand a pressure of 2N-5N without collapsing. After fixing, it is set at a temperature of 45℃-65℃ for 30 minutes to 3 hours.

3. A silk fibroin soft tissue patch, characterized in that, It is prepared using the preparation method described in claim 1 or 2.

4. The application of the silk fibroin soft tissue patch as described in claim 3 in the preparation of soft tissue repair and reinforcement materials, wherein the soft tissue repair and reinforcement materials are used for soft tissue repair in the treatment of chest and abdominal wall defects, abdominal wall hernias, hiatal hernias, diaphragmatic hernias, pelvic floor hernias, inguinal hernias, and tendons and ligaments.

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