Natural silk patch for treating female pelvic floor dysfunction diseases and preparation method thereof

Abstract

The invention discloses a natural silk patch for treating female pelvic floor dysfunction diseases and a preparation method thereof. The preparation method of the natural silk patch comprises the following steps: carrying out warp knitting on natural silk threads, and then carrying out heat setting to obtain the natural silk patch, and the diameter of the natural silk threads is 20 / 22 / 24D. The natural silk patch prepared by the invention can be used as a pelvic organ prolapse patch. The natural silk patch disclosed by the invention is good in mechanical property, capable of meeting mechanical support required by the pelvic floor, good in biocompatibility and light in foreign matter reaction.

Description

Technical Field

[0001] This invention relates to a natural silk patch for treating female pelvic floor dysfunction and its preparation method, belonging to the field of medical technology. Background Technology

[0002] Pelvic organ prolapse (POP) is a common non-fatal condition in middle-aged and elderly women, with an incidence rate of approximately 9.6% in the Chinese female population. Symptomatic POP patients often experience lower back pain, pelvic pain, urinary dysfunction, and sexual dysfunction, frequently requiring further surgery to achieve anatomical repositioning and alleviate symptoms. Traditional surgical methods such as the Brønsted procedure, anterior / posterior vaginal wall repair, and vaginal closure can be used for POP repair, but they suffer from low anatomical repositioning success rates, insignificant symptom relief, and postoperative recurrence of prolapse / symptoms. Transvaginal mesh repair, as an effective surgical tool for POP repair, can enhance surgical repair outcomes, increase patient satisfaction, and reduce postoperative anatomical and / or symptom recurrence; however, it carries surgical risks such as mesh exposure and increased bleeding risk, requiring close monitoring of short-term and long-term postoperative complications. Therefore, it is necessary to develop a new type of mesh material for female pelvic reconstruction. Summary of the Invention

[0003] The purpose of this invention is to provide a natural silk patch for treating female pelvic floor dysfunction, which has excellent mechanical properties, microstructure and biocompatibility, and is suitable for female pelvic reconstruction.

[0004] The method for preparing natural silk patches provided by this invention includes the following steps:

[0005] Natural silk threads are warp-knitted and then heat-set.

[0006] The diameter of the natural silk thread is 20 / 22 / 24D.

[0007] In the above preparation method, the natural silk thread is warped and then warped;

[0008] The DN21 type warping machine is used to wind the single filaments of the natural silk thread onto the warp spool with parallel tension, constant speed, and neatness for use by the warp knitting machine;

[0009] The RS4EL warp knitting machine was used for warp knitting.

[0010] In the above preparation method, the warp knitting conditions are as follows:

[0011] The padding yarn yardage is GB1: 10 / 01 / 10 / 01 / 10 / 01 / 10 / 01 / / , GB2: 10 / 23 / 32 / 23 / 45 / 32 / 23 / 32 / / , GB3: 45 / 32 / 23 / 32 / 10 / 23 / 32 / 23 / / , GB4: 00 / 77 / 00 / 77 / 00 / 77 / 00 / 77 / / ;

[0012] The feed rates are 1600mm / rack, 1650mm / rack, 1650mm / rack and 4100mm / rack respectively.

[0013] In the above preparation method, the heat setting conditions are as follows: using a 350-1000mm heat setting machine at a temperature of 150-200℃ for 6-8 minutes.

[0014] The natural silk patch prepared by this invention has a pore size > 200 μm and an areal density of 43.92 ± 1.9474 g / m³. 2 The porosity is 58.0788%.

[0015] The application of the natural silk patch prepared by this invention as a patch for pelvic organ prolapse is also within the scope of protection of this invention.

[0016] The natural silk patch of this invention has the following beneficial technical effects:

[0017] The patch has good mechanical properties, meeting the mechanical support required by the pelvic floor; the patch has good biocompatibility and mild foreign body reaction. Attached Figure Description

[0018] Figure 1 Scanning electron microscope images of two types of patches, as well as natural silk thread and polypropylene thread.

[0019] Figure 2 The results show the physical properties of polypropylene patches and natural silk patches.

[0020] Figure 3 The results show the tensile strength and elongation at break of polypropylene patches and natural silk patches.

[0021] Figure 4 The results show the test results for the stiffness and Young's modulus of polypropylene patches and natural silk patches.

[0022] Figure 5 The procedure for constructing a rat partial abdominal wall defect surgical model.

[0023] Figure 6 This shows the adhesion of tissues around the patch when the rats were sacrificed 4 weeks after surgery.

[0024] Figure 7The results show the fracture strength and Young's modulus of the patch tissue after sacrifice 4 weeks post-surgery in three groups of rats.

[0025] Figure 8 HE staining after patch implantation (cell nuclei appear blue-purple, n=3).

[0026] Figure 9 MASSON staining after patch implantation (under an optical microscope, collagen fibers appear blue, muscle fibers, natural silk threads, and other tissues such as erythrocytes appear red, and cell nuclei appear blue-black, n=3). Detailed Implementation

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0028] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0029] Example 1: Preparation of the patch

[0030] 1. Warping: Using a DN21 warping machine, natural silk threads (from Rugao Beitefu Thread Co., Ltd.) are wound onto the warp spools with parallel tension, at the same speed, and neatly according to the required number and length of warp threads for use by the warp knitting machine.

[0031] 2. Warp knitting: Use an RS4EL warp knitting machine to knit the mesh according to the process parameters in Table 1 to obtain a silk protein mesh.

[0032] Table 1 Weaving process parameters

[0033] Mesh Comb process Material Quantity of Scriptures density GB1 10 / 01 / 10 / 01 / 10 / 01 / 10 / 01 / / Natural silk 1600 14 GB2 10 / 23 / 32 / 23 / 45 / 32 / 23 / 32 / / Natural silk 1650 14 GB3 45 / 32 / 23 / 32 / 10 / 23 / 32 / 23 / / Natural silk 1650 14 GB4 00 / 77 / 00 / 77 / 00 / 77 / 00 / 77 / / Natural silk 4100 14

[0034] 3. Heat setting: Use a 350-1000mm setting machine to set the shape at 170℃ for 6 minutes.

[0035] Example 2: Physical properties and microstructure of the patch

[0036] I. Experimental Methods

[0037] 1. Electron microscopic observation of patches and braided threads

[0038] 1) Take sterilized and preserved polypropylene patches (Herniamesh Ltd., Italy), natural silk patches, silk threads, natural silk threads (Rugao Beitefu Thread Co., Ltd.), and polypropylene monofilaments (Changzhou Hongxiang Medical Supplies Technology Co., Ltd.), and keep them dry.

[0039] 2) Conductive treatment of the patches: Two types of patches were fixed to a copper plate and sputter-coated with gold for 10 seconds. The surface structure and pore size were observed using a high-performance field emission JSM-7900F scanning electron microscope.

[0040] 2. Physical properties of the patch

[0041] 1) Areal density detection

[0042] Areal density, or the mass of the patch per square meter, was measured using a BA110S analytical balance in this experiment. The sample mass M (in grams) was obtained. The sample dimensions were 100 mm × 100 mm. The areal density of the sample (in g / m²) was... 2 = M / (0.1×0.1), each sample was measured 5 times and the average value was taken.

[0043] 2) Thickness inspection

[0044] Thickness is the distance between the upper and lower opposing surfaces of the patch. This test was conducted according to the national standard GB / T3 820-1997, using a YG141N digital fabric thickness gauge to measure the thickness of the sample. The sample size was 100mm × 100mm, and the presser foot area was 2000±20mm². 2 Place the sample on the reference plate, parallel to the pressure foot of the plate, apply pressure of 1 ± 0.01 kPa, and pressurize for 30 ± 5 s. Record the vertical distance between the two plates, which is the measured sample thickness (in mm). Measure each sample in different areas five times, and take the average value.

[0045] 3) Porosity detection

[0046] Porosity refers to the percentage of the pore area of ​​a patch to its total area under natural conditions. Samples were observed using a JSM-7900F scanning electron microscope. Five different fields of view at 25X were photographed, and the images were imported into Photoshop. After grayscale image processing, the threshold was adjusted to the optimal value to obtain a black and white image. The number of black pixels (Nb) in the pore area and the total number of pixels (Nt) were obtained through histogram processing at 255 levels. The porosity (in %) was calculated as Nb / Nt × 100%. The calculation was performed five times for each sample, selecting different regions, and the average result was taken.

[0047] 4) Fracture strength test

[0048] Breaking strength is the maximum force exerted on a patch under specified conditions until it breaks. An AGS-X electronic universal testing machine was used to test the tensile properties of the test patch. The test conditions followed the national standard GB / T3923.1-1997: the machine's lifting speed was set to 100 mm / min, the clamping distance to 200 mm, and the pre-tension to 1 N. Breaking strength is the maximum force (in N) recorded when the test sample breaks during the tensile test. Test samples with dimensions of 50 mm × 300 mm were cut from both the longitudinal and transverse sides of the specimen, and each side was tested five times. The average value of the results was taken.

[0049] 5) Elongation at break test

[0050] Elongation at break is the ratio of the elongation of the patch at maximum force during tension to its initial length before tensioning. An AGS-X electronic universal testing machine was used to test the tensile properties of the test patch. The machine's lifting speed was set to 100 mm / min, the clamping distance to L (200 mm), and the pre-tension to 1 N. The test sample was measured as the increment of its length ΔL (in mm) corresponding to the breaking strength during the tensile process. Elongation at break (%) = ΔL / L × 100%. Test samples of 50 mm × 300 mm were cut from both the longitudinal and transverse sides of the sample, and each sample was tested five times. The average value of the results was taken.

[0051] 6) Stiffness testing

[0052] Stiffness is the ability of a patch to resist elastic deformation when subjected to force. The experimental method is the same as in 5). The force F (in N) acting on the patch during the process of stretching the sample to fracture is measured. The deformation caused by the force is the elongation increment ΔL (in cm) of the sample. Stiffness K (in N / cm) = F / ΔL. The sample size is 50mm × 10mm. Samples are cut 5 times each in the longitudinal and transverse directions of the sample, and the average value is taken.

[0053] 7) Young's modulus testing

[0054] Young's modulus is a physical quantity that describes the ability of a patch to resist deformation. The experimental method is the same as in 6), A (unit: mm). 2 The cross-sectional area of ​​the test sample is given by , L (in mm) is the length of the sample (i.e., the clamping distance), F (in N) is the stress on the test sample during the stretching process to fracture, ΔL (in mm) is the elongation increment of the sample corresponding to stress F, and E (in N / mm²) is the Young's modulus. 2 = (F·L) / (A·△L). The test sample size is 50mm×300mm, and the test is performed 5 times. The average value of the result is taken.

[0055] II. Experimental Results

[0056] 1. Electron microscopic observation results of patches and braided threads

[0057] After being sprayed with gold, the structure and surface of the two types of patches were observed under low and high magnification using a scanning electron microscope. It can be observed that the polypropylene threads used in the polypropylene patches are all single polypropylene filaments, while the single strand of natural silk thread used in the natural silk patch is made by condensing and twisting dozens of natural silk monofilaments.

[0058] The polypropylene patch has uniform and regular mesh openings, with diameters mainly ranging from 800 μm to 1200 μm. Figure 1e). The mesh diameter of natural silk patches ranges from 300μm to 1500μm. Figure 1 a) Primarily large pore sizes of 500μm and above.

[0059] The warp knitting process using natural silk threads makes the structure of the natural silk patch more complex and three-dimensional, the knots more secure, and less prone to deformation. Figure 1 a, Figure 1 b and Figure 1 c). Natural silk is coated with a layer of sericin, approximately 60 μm in diameter. Figure 1 d) Some natural silk threads have an uneven, burr-like structure on their surface due to the removal of silk fibers from the silk. Figure 1 c).

[0060] The polypropylene single-line surface used in polypropylene patches is smooth and burr-free. Figure 1 f and Figure 1 g), the diameter of the monopropylene wire is approximately 80 μm ( Figure 1 h).

[0061] 2. Analysis of the physical and mechanical properties of the patch

[0062] 1) Surface density

[0063] Areal density refers to the mass of the patch per square meter. A comparison of the areal densities of the two types of patches is shown in Table 2. Figure 2 a) Polypropylene patch < natural silk patch, the difference was statistically significant (p<0.001).

[0064] 2) Thickness

[0065] Comparison of the thicknesses of the two types of patches (Table 2) Figure 2 b): Natural silk patch < polypropylene patch, and the difference was statistically significant (p<0.01).

[0066] 3) Porosity

[0067] Comparison of porosity between the two types of patches (Table 2) Figure 2 c) The porosity of natural silk patches was slightly greater than that of polypropylene patches, but the difference was not statistically significant (p>0.05). The porosity of both natural silk patches and polypropylene patches was greater than 50%.

[0068] Table 2 Physical properties of the two types of patches (x±SD)

[0069]

[0070] 4) Fracture strength

[0071] The fracture strength measurement results for the two types of patches are as follows (Table 3). Figure 3Polypropylene patches, due to their uniform and stable rhomboid structure, do not exhibit longitudinal or transverse strength differences. The longitudinal breaking strength of both types of patches is >30N, with the polypropylene patch showing a higher strength than the natural silk patch, but the difference is not statistically significant (p = 0.2116). The transverse breaking strength of natural silk is lower, showing a statistically significant difference compared to the polypropylene patch (p < 0.05), but not a statistically significant difference compared to its own longitudinal breaking strength (p = 0.1282).

[0072] 5) Elongation at break

[0073] Longitudinal and transverse elongation at break of the two types of patches (Table 3, Figure 3 The order of elongation at break was: polypropylene patch < natural silk patch (longitudinal direction < natural silk patch (transverse direction)). There was no statistically significant difference in the longitudinal elongation at break between the natural silk patch and its transverse elongation at break (p = 0.0527). There was no statistically significant difference in the longitudinal elongation at break between the polypropylene patch and the natural silk patch (p = 0.9987), but a statistically significant difference was found between the polypropylene patch and the natural silk patch (p < 0.05).

[0074] Table 3 Mechanical properties of the two types of patches (x±SD)

[0075]

[0076] 6) Stiffness

[0077] Stiffness is the ability of a patch to resist elastic deformation under stress; the greater the stiffness, the less likely the patch is to undergo elastic deformation under stress. A comparison of the longitudinal stiffness of the two types of patches is shown in Table 4. Figure 4 The longitudinal stiffness of polypropylene patches was significantly greater than that of natural silk patches, which was greater than that of natural silk patches in the transverse direction. The differences were statistically significant (p<0.05), while there was no statistically significant difference in longitudinal stiffness between the two types of patches (p=0.7793).

[0078] Table 4. Stiffness and Young's modulus (x±SD) of the two types of patches

[0079]

[0080] 7) Young's modulus

[0081] Young's modulus and stiffness are both physical quantities that describe the ability of a patch to resist deformation. A comparison of the Young's modulus of the two types of patches is shown in Table 4. Figure 4 The results showed that the longitudinal modulus of natural silk patches was significantly higher than that of polypropylene patches, with the latter being the highest in the longitudinal direction (p<0.05). There was no significant difference in the transverse modulus between natural silk patches and polypropylene patches (p=0.0569).

[0082] III. Comparison of Experimental Results

[0083] 1. Comparison of the physical properties of the two types of patches

[0084] Infection after patch implantation can lead to patch exposure or erosion. An ideal POP (Pelvic Floor Organization) patch requires biomechanical properties, biocompatibility, and anti-infection capabilities suitable for pelvic floor tissues. Multiple studies have shown that lower patch areal density and larger pore size result in a lower probability of infection, less noticeable foreign body reaction, and less scar bridging. However, excessively low areal density can also lead to patch folding and difficulties in surgical manipulation. Therefore, it is necessary to explore a suitable range for patch areal density to ensure optimal mechanical properties and biocompatibility.

[0085] Besides the pore diameter of the patch, the spacing between the individual filaments is also a crucial factor influencing postoperative complications. Compared to monofilament mesh patches, multifilament patches implanted in the abdominal wall of rats exhibit more pronounced and persistent inflammatory and fibrotic responses. If the pores are very small (<10μm), leukocytes (9–15μm) and macrophages (16–20μm) cannot pass through and cannot participate in the immune response against infection, while bacteria (<2μm) can perfectly colonize the small pores, increasing the risk of infection. Furthermore, patch diameters <10μm or multifilament materials may lead to postoperative infection; while patch pore diameters >100μm facilitate the migration of macrophages and leukocytes, reducing the risk of infection and patch-related complications, and also allow fibroblast adhesion and migration, generating more collagen fibers, enabling the patch to better integrate into the host tissue. The size of the patch pores not only affects the migration of bacteria and inflammatory cells and the ingrowth of new tissue but also the flexibility of the patch. Patches with smaller pores are harder, increasing the risk of patch erosion, postoperative pelvic pain, and functional impairment. Large-aperture, monofilament, and lightweight patches are more suitable for POP repair surgery.

[0086] Patches are typically classified as lightweight (<40g / m²) based on their weight per unit area. 2 ), medium weight type (40-85g / m 2 ) and heavy type (>85g / m 2 The results of this invention show that the areal density of natural silk patches is slightly greater than that of polypropylene patches, but the areal density of both is less than 40 g / m². 2 Both are lightweight patches. Electron microscopy images show that the three-dimensional structure of natural silk patches is more complex than that of polypropylene patches, suggesting that they are more suitable for cell adhesion and migration, and are conducive to the ingrowth of new tissue.

[0087] In addition, most of the pores in both types of patches are larger than 200 μm, allowing inflammatory cells to migrate in and new tissue to grow in. However, the natural silk patch uses multifilament silk threads, which have some small pores smaller than 100 μm between the braided threads. This poses a risk of infection after the patch is implanted in the body, and further in vivo experiments are needed to study the biocompatibility of the patch.

[0088] 2. Comparison of the mechanical properties of the two types of patches

[0089] Increased pelvic and abdominal pressure, along with weakened strength of the abdominal wall and pelvic floor supporting muscles and fascia, are the main causes of abdominal wall hernias and post-operative pelvic inflammatory disease (POP). In a resting supine position, intra-abdominal pressure is approximately 4 mmHg, while in a resting upright position it is 5-6 times higher. During occasional events causing increased abdominal pressure, such as coughing, the maximum measurable intra-abdominal pressure can reach 180 mmHg, and the stress acting on the abdominal wall muscles and repair mesh can reach 16 N / cm. In contrast, the pressure borne by the repair mesh after abdominal wall hernia repair in a resting state is 2 N / cm.

[0090] After pelvic floor prolapse (POP) patients undergo patch repair surgery, pelvic and abdominal pressures also act on the pelvic floor support tissues and the surgical patch. Currently, there is a lack of data regarding the required mechanical properties of patches for POP repair; the generally accepted approach is that pelvic floor patches should have greater mechanical properties than those required for abdominal defect repair patches.

[0091] However, if the implanted patch is too stiff, the surrounding tissues may atrophy due to disuse under the stress barrier effect of the patch, leading to patch exposure and erosion. Feola et al. implanted patches of different stiffness into rhesus monkeys for sacral vaginal fixation after hysterectomy and found that the mechanical properties of the macaque's vaginal tissue deteriorated most severely after implantation of the patch with the highest stiffness, with the most significant decrease in vaginal contractility and vaginal tissue stiffness. Liang et al.'s in vivo experiments on macaques also support this conclusion. The experiments showed that the implantation of high-stiffness, hard patches significantly thinned the vaginal smooth muscle layer, increased apoptosis of cells around the patch, and reduced the content of collagen and elastin, all due to the stress shielding effect.

[0092] The results of this invention show that the natural silk patch prepared in this invention has a similar breaking strength to the polypropylene patch used clinically. However, the natural silk patch has lower stiffness and a softer texture, resulting in minimal strength difference compared to normal pelvic floor tissue while meeting the required mechanical support for the pelvic floor. It is speculated that the natural silk patch integrates better with surrounding tissues after implantation, forming a patch-tissue complex that is more suitable for pelvic floor support.

[0093] The above analysis shows that the microstructure of natural silk patches exhibits a rough surface and multifilament weaving, resulting in a more complex three-dimensional structure than polypropylene patches. The pore size is >200μm, which is beneficial for cell adhesion and migration, facilitating the ingrowth of inflammatory cells and new tissue. The tensile strength, stiffness, Young's modulus, and other mechanical properties of natural silk patches meet or even exceed the mechanical requirements of clinically used polypropylene patches, making them an ideal choice for pelvic floor reconstruction patches.

[0094] Example 3: Biocompatibility Study of the Patch

[0095] I. Experimental Methods

[0096] (1) Patch preparation

[0097] 1) Prepare two types of patches into separate 1×2cm bags for individual packaging;

[0098] 2) Sterilize the packaged patches using ethylene oxide and store them in a fume hood for at least two weeks before use.

[0099] (2) Animal experiments

[0100] 1) Grouping

[0101] Twenty-seven female SD rats were divided into three groups: a polypropylene patch group (n=9), a natural silk patch group (n=9), and a blank control group (n=9). All rats underwent partial abdominal wall muscle defect surgery.

[0102] 2) Construction of a rat model of partial abdominal wall muscle defect and surgical repair of the defect.

[0103] Rats were anesthetized with isoflurane at a rate of 5 cc / min, and maintained at a rate of 2 cc / min after satisfactory anesthesia. The limbs were immobilized, and the abdominal hair was shaved. The abdominal skin was disinfected three times with 75% alcohol. A 3 cm longitudinal incision was made in the lower abdomen, and the skin and subcutaneous tissue were bluntly dissected. The abdominal wall muscle layer was longitudinally incised 1 cm on each side of the linea alba, 2 cm to the left and right. The internal oblique and transversus abdominis muscles were bluntly dissected, and parts of the external and internal oblique muscles were removed. A 1×1 cm partial abdominal wall muscle defect was constructed on each side of the linea alba, preserving the transversus abdominis muscle and peritoneum.

[0104] In the experimental group, a 1×2cm patch was placed over the defect, and the edges of the patch were secured to the surrounding tissue of the abdominal wall defect with 5-0 absorbable sutures without tension. In the blank control group, a defect model was constructed, followed by compression hemostasis and four-point suture at the same location. The incision was sutured layer by layer and disinfected with 75% alcohol. Intraoperative bleeding was 1-2ml, and postoperative analgesic jelly was administered for comfort.

[0105] 3) Draw materials

[0106] Rats were fed routinely after surgery and sacrificed at 1, 4, and 12 weeks post-surgery, with 3 rats from each group sacrificed each time. After sacrifice, the implanted patches and surrounding tissues from the experimental group and the abdominal wall tissue with surgical sutures from the control group were removed from both sides. The mechanical properties of tissues from one side of the same rat were tested immediately after removal, while the tissues from the other side were fixed by immersion in 4% paraformaldehyde for 24 hours.

[0107] During tissue sampling, the adhesion between the surgical site and subcutaneous tissue was scored according to the Walker scoring criteria: 0 points: no adhesion; 1 point: adhesion area <25%, can be bluntly separated with a small force; 2 points: adhesion area ≥25%, can be bluntly separated with a large force; 3 points: tight adhesion, sharp separation is required.

[0108] (3) Testing of the mechanical properties of the patch

[0109] The tensile properties of the test tissue were tested using an AGS-X type electronic universal testing machine. Due to the limited size of the tissue, the test sample size was 20mm × 10mm. The test conditions were adjusted according to the standard GB / T 3923.1-1997. The tensile speed of the testing machine was set to 10mm / min, the clamping distance was 10mm, and the pre-tension was 1N. During the tensile test of the test sample, the maximum force recorded when the sample broke was the breaking strength (unit N). The breaking strength is the absolute value that represents the strength of the material.

[0110]

[0111] (4) Tissue embedding and fixation

[0112] 1) Sampling: After fixing fresh tissue with 4% paraformaldehyde for more than 24 hours, the tissue block is taken out and the tissue at the target site is trimmed flat with a scalpel in a fume hood. The trimmed tissue is then placed in a marked dehydration box.

[0113] 2) Dehydration: 5% alcohol for 4h → 85% alcohol for 2h → 90% alcohol for 2h → 95% alcohol for 1h → anhydrous ethanol I for 30min → anhydrous ethanol II for 30min → benzene for 5-10min → xylene I for 5-10min → xylene II for 5-10min → paraffin I for 1h → paraffin II for 1h → paraffin III for 1h.

[0114] 3) Embedding: Place the wax-impregnated tissue in the embedding machine for embedding, cool it on a -20°C freezing stage, and remove the wax block from the embedding frame and trim the wax block after the wax has solidified.

[0115] 4) Sectioning: Place the trimmed wax block on a paraffin microtome and section it to a thickness of 3-4 μm. Float the sections on 40°C warm water in a slide spreader to flatten the tissue, attach them to anti-detachment glass slides and number them, then place them in a 60°C oven to bake. After the water has dried and the wax has melted, remove them and store them at room temperature for later use.

[0116] (5) HE staining

[0117] Two sections were randomly selected from each tissue block for HE staining. Specific steps included:

[0118] 1) Baking slices: Bake slices in a 65℃ oven for 1 hour.

[0119] 2) Dewaxing to water: Place the sections in xylene I for 20 min → xylene II for 20 min → anhydrous ethanol I for 10 min → anhydrous ethanol II for 10 min → 95% ethanol for 5 min → 90% ethanol for 5 min → 80% ethanol for 5 min → 70% ethanol for 5 min → wash with distilled water for 1 min.

[0120] 3) Hematoxylin staining of cell nuclei: Place the slices in Harris hematoxylin staining for 3-8 minutes, rinse with tap water for 1 minute, differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with tap water, return to blue with 0.6% ammonia water, and rinse with running water.

[0121] 4) Eosin staining of cytoplasm: Immerse the slide in eosin staining solution for 1-3 minutes, then wash with tap water for 1 minute.

[0122] 5) Dehydration and mounting: Place the sections in 95% ethanol I for 5 min → 95% ethanol II for 5 min → anhydrous ethanol I for 5 min → anhydrous ethanol II for 5 min → xylene I for 5 min → xylene II for 5 min to dehydrate and clear them. Remove the sections from the xylene and let them dry slightly before mounting them with neutral resin.

[0123] 6) Interpretation: Determine the results under the microscope, and analyze the images.

[0124] Staining results: The cell nuclei appeared blue-purple.

[0125] 7) Inflammatory cell count

[0126] Inflammatory cell count: HE slides were placed under a 400× optical microscope, and five fields of view were counted for each slide. The proportion of neutrophils in each field of view was calculated and compared, and the average value of the results was taken.

[0127] (6) Masson staining

[0128] Masson staining is one of the main methods for visualizing fibers in tissues, primarily used to identify collagen fibers and muscle fibers. To identify the components of newly formed tissue around the patch, two sections of paraffin-embedded tissue blocks were randomly selected at 1 week, 4 weeks, and 12 weeks post-surgery for Masson staining. Specific procedures were performed according to the kit instructions.

[0129] 1) Baking slices: Bake slices in a 65℃ oven for 1 hour.

[0130] 2) Dewaxing to water: Place the sections in xylene I for 20 min → xylene II for 20 min → anhydrous ethanol I for 10 min → anhydrous ethanol II for 10 min → 95% ethanol for 5 min → 90% ethanol for 5 min → 80% ethanol for 5 min → 70% ethanol for 5 min → wash with distilled water for 1 min.

[0131] 3) Hematoxylin staining of cell nuclei: Weigert's iron hematoxylin staining kit for 5 min, rinse with tap water, differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with tap water for 1-3 min, blue with 1% ammonia water for 30 s, rinse with tap water for 1-3 min.

[0132] 4) Ponceau S staining: Stain with Ponceau S acid fuchsin solution from the Masson staining kit for 5-10 minutes, then rinse quickly with distilled water.

[0133] 5) Phosphomolybdic acid treatment: Add phosphomolybdic acid aqueous solution from the Masson staining kit and stain for about 3-5 minutes.

[0134] 6) Aniline blue staining: Counterstain with aniline blue solution from the Masson staining kit for 5 min, then differentiate with 1% glacial acetic acid solution for about 1 min.

[0135] 7) Dehydration and mounting: Place the sections in 95% alcohol I for 5 min → 95% alcohol II for 5 min → anhydrous ethanol I for 5 min → anhydrous ethanol II for 5 min → xylene I for 5 min → xylene II for 5 min to dehydrate and clear them. Remove the sections from the xylene and let them dry slightly before mounting them with neutral resin.

[0136] 8) Interpretation: Determine the results under the microscope, and analyze the images.

[0137] Staining results: Collagen fibers appear blue; muscle fibers, cellulose, and other tissues such as erythrocytes appear red; cell nuclei appear blue-black.

[0138] (7) Statistical analysis

[0139] The above data were entered into GraphPad Prism Version 8.2.1 software. The Kolmogorov-Smironov test was used to assess the normality of the data distribution. Data conforming to a normal distribution are expressed as mean ± standard deviation (x ± SD). One-way ANOVA was used for comparisons between groups, with p < 0.05 considered statistically significant.

[0140] II. Experimental Results

[0141] 1. Construction of animal models

[0142] Female SD rats were randomly divided into three groups, all of which underwent partial abdominal wall muscle defect surgery: a blank control group, a polypropylene patch group, and a natural silk patch group. A rat model of partial abdominal wall muscle defect was successfully established. Figure 5 The procedures involved simple surgical sutures, insertion of polypropylene patches, and insertion of natural silk patches, respectively. All animals survived after surgery and were kept in routine care. No problems such as animal infection, patch exposure, or patch erosion were observed.

[0143] In the blank control group, only a small amount of adhesion formed at the surgical site 1 week after surgery, with an adhesion area of ​​<25%, and 4 weeks after surgery ( Figure 6 a) At this time, most of the surgical site is covered by fibrous connective tissue, but it can be bluntly dissected.

[0144] One week post-surgery, the silk fibroin patch group had a moderate amount of fibrous connective tissue covering the patch above and around it, with 50% > adhesion area > 25%, all of which could be bluntly separated; four weeks post-surgery ( Figure 6 b) The fiber wrapping increases compared to before, but it can still be bluntly separated.

[0145] One week post-surgery, the polypropylene patch group showed only a small amount of connective tissue above and around the material, with adhesion area <25%, allowing for blunt dissection. Four weeks post-surgery, fibrous encapsulation increased. Figure 6 c) The patch is covered with a large amount of fibrous connective tissue above and around it, and is closely adhered to the subcutaneous tissue, requiring sharp dissection for sampling.

[0146] 2. Results of force measurement in rat tissue

[0147] 1) Differences in fracture strength among the three groups at 4 weeks post-surgery (Table 5, Figure 7 )

[0148] 1. Four weeks post-surgery, the breaking strength of the blank control group was significantly lower than that of the polypropylene patch group (p<0.0001); and slightly lower than that of the natural silk patch group (p<0.05).

[0149] 2. Four weeks post-surgery, the breaking strength of the natural silk group was significantly lower than that of the polypropylene patch group, and the difference was statistically significant (p<0.0001).

[0150] Table 5 Comparison of patch tissue breaking strength and Young's modulus in three groups of rats 4 weeks after surgery (x±SD)

[0151]

[0152] 2) Differences in Young's modulus among the three groups at 4 weeks post-surgery (Table 5, Figure 7 )

[0153] 1. Four weeks post-surgery, the breaking strength of the blank control group was significantly lower than that of the polypropylene group (p<0.05); it was also lower than that of the natural silk patch group (p=0.0531).

[0154] 2. Four weeks post-surgery, the breaking strength of the natural silk patch group was slightly lower than that of the polypropylene patch group, but the difference was not statistically significant (p = 0.7868).

[0155] 3. Analysis of inflammatory response and tissue regeneration after patch implantation

[0156] (1) Analysis of HE staining results

[0157] 1. In the blank control group, a small number of inflammatory cells, mainly lymphocytes, were observed at 1 and 2 weeks post-surgery. By 4 weeks post-surgery, the number of inflammatory cells gradually decreased, with only a small amount of loosely arranged connective tissue remaining. Figure 8 ).

[0158] 2. In the polypropylene patch group, a large number of inflammatory cells, mainly neutrophils, were observed on the surface of the polypropylene monofilament at 1 week postoperatively. At 2 and 4 weeks postoperatively, the neutrophil count decreased significantly, and a small number of foreign body giant cells were observed. At 4 weeks postoperatively, there was more connective tissue surrounding the patch compared to 1 week postoperatively. Figure 8 ).

[0159] 3. In the natural silk patch group, more neutrophils were observed around the material at 1 and 2 weeks post-surgery, and more foreign body giant cells were observed compared to the polypatch group. Figure 8 The patch was surrounded by a significant amount of connective tissue; four weeks post-surgery, the connective tissue gradually grew into the natural silk patch and wrapped around the natural silk filaments in a regular pattern. Widening of the gaps between the natural silk filaments and the surrounding connective tissue were clearly observed, indicating in vivo degradation of the natural silk. Figure 8 ).

[0160] (2) Analysis of Masson staining results

[0161] 1. In the blank control group, a small amount of collagen hyperplasia was observed one week after surgery. Collagen gradually increased at two and four weeks post-surgery, and a significant collagen regeneration layer with a denser structure was visible at four weeks. Figure 9 ).

[0162] 2. In the polypropylene patch group, only a small amount of collagen regeneration was observed one week post-operation. Collagen fiber increase occurred at two weeks post-operation; at four weeks post-operation, the amount of collagen fiber was significantly increased compared to one and two weeks post-operation, with a small amount of newly formed collagen fibers ingrained into the patch. Figure 9 ).

[0163] 3. In the natural silk patch group, more collagen fiber regeneration was observed above and below the patch one week post-operation, significantly more than in the blank control group and the polypropylene patch group. At two weeks post-operation, newly formed collagen fibers were seen growing into the interior of the patch in the natural silk patch group, and at four weeks post-operation, the newly formed collagen fibers tightly wrapped around the patch, forming a tight connection.

[0164] III. Analysis of Experimental Results

[0165] 1. Foreign body reaction after patch implantation

[0166] Any implanted material will elicit a host response to the foreign body, known as foreign body reaction (FBR). This response depends on the material's properties and the host's susceptibility. FBR is unavoidable. An ideal pelvic floor surgical patch should possess good biocompatibility, not only providing support but also integrating into the implanted tissue without causing excessive foreign body reactions. FBR mainly includes five stages: protein adsorption, acute inflammatory response, chronic inflammatory response, foreign body giant cell formation, and fibrous capsule formation.

[0167] (1) Protein adsorption

[0168] When the material is implanted into the body, an immediate interaction occurs between the trauma and the blood material. Within seconds, proteins in the plasma adsorb onto the material, forming a very sparse temporary protein matrix of 2-5 nm. This matrix is ​​mainly composed of fibrin, which forms thrombi with specific regional differences.

[0169] (2) Acute inflammatory response

[0170] The acute inflammatory response, characterized by infiltration of polymorphonuclear leukocytes (PMNs) (primarily neutrophils, with a small number of eosinophils and basophils) and mast cells, is the second stage of FBR. It typically lasts from a few hours to several days, usually subsiding within a week, and may evolve into chronic inflammation. Cytokines released by PMNs can influence the characteristics and extent of subsequent inflammatory cell recruitment and activation, thus affecting the macrophage phenotype during the chronic inflammatory phase. Brodbeck et al.'s research suggests that the initial adhesion of monocytes to the implant material is transient, serving as a signal to recruit and activate lymphocytes. After lymphocytes release cytokines, monocytes persist and differentiate into macrophages, eventually differentiating into foreign body giant cells.

[0171] (3) Chronic inflammatory response

[0172] Chronic inflammatory responses typically occur 2–3 weeks after biomaterial implantation, and are characterized by the infiltration of lymphocytes and monocytes. Monocytes differentiate into macrophages with different phenotypes under the influence of various cytokines. The main macrophages exhibit a wound-healing phenotype, producing various growth factors to promote cell proliferation and angiogenesis, and recruiting fibroblasts to form fibrous capsules that encapsulate the implant, separating it structurally and functionally from the host tissue.

[0173] Madden et al. found that the pore size of the implant also significantly affected the chronic inflammatory trend of fibroblast-like scaffolds (FBRs). Cardiac implantation with cell-free scaffolds with pore sizes of 30–40 μm resulted in the discovery of more infiltrative macrophages, with a higher proportion of M2 macrophages, guiding rapid angiogenesis and anti-fibrotic responses, and exhibiting the best material-tissue integration. Implant rigidity can also influence the inflammatory response trend; the mismatch between rigid implant materials and the lower tissue stiffness surrounding the implantation site may lead to higher mechanical stimulation and increased inflammation. If a biodegradable material is implanted, the inflammatory phase is prolonged, multinucleated giant cells appear, and collagen production trends are altered.

[0174] (4) Formation of foreign giant cells

[0175] The formation of foreign body giant cells (FBGs) is considered a hallmark of febrile inflammatory bowel disease (FBR), distinguishing it from other chronic inflammatory conditions. FBGC formation is a process of macrophage fusion, typically resulting in large, multinucleated giant cells with diameters ranging from tens to hundreds of micrometers. Furthermore, FBGCs persist as long as the implant material, such as non-absorbable material, remains. Studies have shown that implants with rough surfaces, large surface volumes, and high granularity and porosity induce the formation of more FBGCs than implants with smooth, flat surfaces.

[0176] (5) Fibrosis

[0177] In most cases, the ideal outcome is that the implanted biomaterial integrates completely into the surrounding tissue after slow degradation. However, if it cannot be rapidly eliminated through phagocytosis or degraded in vivo, macrophages and FBGCs accumulate around the invaded tissue or on the surface of the foreign body, forming fibrous capsules to isolate and prevent its spread to other parts of the body. The formation of fibrous capsules is influenced by various pro-fibrotic and pro-angiogenic growth factors, which are mainly secreted by M2 macrophages, but can also be secreted by other cell types such as keratinocytes and fibroblasts.

[0178] The influence of implant surface morphology, particularly porosity, on fibrous capsule formation has been extensively studied. Numerous studies have concluded that implant surface morphology affects early pro-inflammatory cytokine production and monocyte / macrophage transcription. Increased implant porosity can reduce the thickness of the formed fibrous capsule, decrease fibrosis and implant material encapsulation, and allow for better integration into the tissue.

[0179] The results of this study show that the natural silk patch group had the highest neutrophil count and the most severe acute inflammatory response at 1 week post-surgery; however, it also showed the earliest and most numerous foreign body giant cells compared to the polypropylene patch group. By 4 weeks post-surgery, the number of neutrophils in the natural silk patch group was significantly reduced, with only a small number of lymphocytes surrounding the patch. The polypropylene patch group showed less neutrophil infiltration in the early stages, but this did not decrease at 2 and 4 weeks.

[0180] Four weeks post-surgery, the polypropylene patch group showed more severe adhesions, requiring sharp dissection during sampling. Tissue staining revealed a dense fibrous encapsulation around the polypropylene. In contrast, the natural silk patch group exhibited less adhesion to the tissue, allowing for blunt dissection during sampling. Tissue staining showed that collagen fibers gradually grew into the patch and encapsulated the natural silk monofilaments.

[0181] These differences in inflammatory responses and newly formed tissue are related to the material and structural design of the patch itself. The large diameter, non-degradable nature, and low reactivity of polypropylene patch monofilaments allow inflammatory cells and newly formed collagen fibers to surround the patch, inducing the formation of fibrous plates and separating the tissue from the material. In contrast, the in vivo degradability of natural silk and its small-diameter monofilaments induce phagocytosis by macrophages and the formation of foreign body giant cells. Natural silk can gradually degrade under these influences. The small pores between the monofilaments within the braid of the natural silk patch allow for the infiltration of inflammatory cells and rapid ingrowth of collagen fibers, enabling the patch to quickly integrate with the surrounding tissue.

[0182] The lower breaking strength of the natural silk patch group at 4 weeks post-surgery compared to the polypropylene group is attributed to the degradation of the natural silk and the destruction of the patch structure. However, it is higher than that of the blank control group and is sufficient to provide the necessary support for the pelvic floor structure. Long-term in vivo degradation, tissue ingrowth patterns and distribution trends, and changes in biomechanical properties require observation over a longer experimental period.

[0183] 2. Occurrence of patch-related complications

[0184] Although fibrosis is necessary during the inflammatory phase, it can also lead to patch-related complications such as patch breakage, erosion, and adhesion formation.

[0185] (1) Patch cracking and erosion

[0186] After surgical patch implantation, a foreign body reaction occurs. The persistent presence of the implant leads to a chronic foreign body response. Macrophages fail to engulf the device and fuse into foreign body giant cells; fibroblasts deposit excessive collagen fibers, eventually encapsulating the surgical patch and forming fragile fibrotic scars. Exacerbated foreign body reaction, persistent inflammation, and subsequent fibrosis are considered the main causes of polypropylene patch rupture. To address this issue, the tissue response to the implanted material must be directed towards the restoration and regeneration of normal tissue, rather than excessive fibrosis—a process also known as structural remodeling. Structural remodeling can be improved by increasing angiogenesis and reducing inflammation.

[0187] (2) Adhesion

[0188] The recommended and widespread use of laparoscopic pelvic floor reconstruction meshes has brought attention to mesh-related adhesions, which are caused by fibrinous exudates following any form of trauma. These exudates form temporary adhesions until the fibrinolytic system absorbs the fibrin. When ischemia, inflammation, or foreign bodies (such as meshes) are present, fibrin absorption can be delayed, or even lead to tissue adhesions.

[0189] When patches are placed near the intestines, all patches will adhere, the extent of which depends on the pore size, filament structure, and surface area of ​​the patch. Heavier patches induce a strong fibrotic reaction, ensuring strong adhesion to surrounding tissues, but also resulting in denser adhesion. Conversely, microporous patches do not allow tissue to grow inwards and have a very low risk of adhesion, but the patch itself does not adhere firmly to the abdominal wall.

[0190] (3) Infection

[0191] The risk of infection depends primarily on the type of filament and pore size used. Microporous patches pose a higher risk of infection because macrophages and neutrophils cannot enter small pores (<10 μm), allowing only bacteria (<1 μm) to survive within the mesh. Similar issues can arise with multifilament patches. Therefore, the meshes with the lowest risk of infection are those made of monofilament with pores larger than 75 μm, eliminating the need to remove these pores to prevent infection.

[0192] The two types of patches used in this study both have a main pore size >200μm, which meets the requirements for immune inflammatory response and new tissue ingrowth. However, the presence of small pores between the monofilaments of natural silk may increase the risk of infection. The rigidity of the polypropylene patch is significantly greater than that of the natural silk patch and the mechanical requirements of the pelvic floor, posing risks such as disuse atrophy of surrounding tissues and patch exposure. Up to 4 weeks post-surgery, no complications such as infection, patch exposure, or erosion were observed in the experimental rats.

[0193] Based on the above analysis, the following conclusions can be drawn:

[0194] 1. Due to the presence of polypropylene monofilaments, by 4 weeks post-surgery, the mechanical properties of the polypropylene patch group far exceeded those of the natural silk patch group and the blank control group; the natural silk patch degraded and integrated into the surrounding tissues after implantation, and the resulting patch tissue complex had a fracture strength and stiffness more similar to the adjacent tissues, which could meet the mechanical requirements of the pelvic floor.

[0195] 2. The natural silk patch group induced more new collagen production compared to the polypropylene patch group and the blank control group. Collagen fibers rapidly grew into the patch and wrapped around the natural silk monofilaments, resulting in better integration of the patch with the tissue compared to the polypropylene patch.

Claims

1. A method for preparing a natural silk patch, characterized in that: Includes the following steps: Natural silk threads are warp-knitted and then heat-set. The diameter of the natural silk thread is 20 / 22 / 24D.

2. The preparation method according to claim 1, characterized in that: The natural silk thread is warped and then warped; The RS4EL warp knitting machine was used for warp knitting.

3. The preparation method according to claim 1 or 2, characterized in that: The conditions for warp knitting are as follows: The padding yarn yardage is GB1: 10 / 01 / 10 / 01 / 10 / 01 / 10 / 01 / / , GB2: 10 / 23 / 32 / 23 / 45 / 32 / 23 / 32 / / , GB3: 45 / 32 / 23 / 32 / 10 / 23 / 32 / 23 / / , GB4: 00 / 77 / 00 / 77 / 00 / 77 / 00 / 77 / / ; The feed rates are 1600mm / rack, 1650mm / rack, 1650mm / rack and 4100mm / rack respectively.

4. The preparation method according to claim 1 or 2, characterized in that: The heat setting conditions are as follows: using a 350-1000mm heat setting machine at a temperature of 150-200℃ for 6-8 minutes.

5. A natural silk patch prepared by the method according to any one of claims 1-4.

6. The natural silk patch according to claim 5, characterized in that: The natural silk patch has a pore size > 200 μm and an areal density of 43.92 ± 1.9474 g / m³. 2 The porosity is 58.0788%.

7. The use of the natural silk patch as described in claim 5 or 6 as a patch for pelvic organ prolapse.

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