CN114870081B - Warp-knitted silk fibroin patch for the treatment of female pelvic floor dysfunction - Google Patents

Abstract

The invention discloses a warp-knitted silk fibroin patch for treating female pelvic floor dysfunction diseases. The preparation method of the warp-knitted silk fibroin patch includes the following steps: warp-knitting the silk fibroin thread, and then heat-setting to obtain the silk fibroin thread; the diameter of the silk fibroin thread is 120-125 µm. The silk fibroin patch prepared by the method of the invention has a pore size of more than 200 µm, an areal density of 51-53 g/m ² and a porosity of 39-45%. The silk fibroin patch prepared by the invention can be used as a pelvic organ prolapse patch. The silk fibroin patch of the present invention can reduce the foreign body reaction after the patch is implanted, and has good biocompatibility; the silk fibroin patch of the present invention has a softer texture, and is compatible with the normal pelvic floor on the basis of satisfying the mechanical support required by the pelvic floor. Tissue strength gaps are minimal and ideal for pelvic floor reconstruction patches.

Description

Warp-knitted silk fibroin patch for the treatment of female pelvic floor dysfunction diseases

technical field

The invention relates to a warp-knitted silk fibroin patch for treating female pelvic floor dysfunction diseases, and belongs to the technical field of medicine.

Background technique

Pelvic organ prolapse is a common non-fatal disease among middle-aged and elderly women. The incidence rate of female population in my country is about 10%. Symptomatic POP patients are often accompanied by problems such as backache, pelvic pain, abnormal urination function, and sexual dysfunction. Surgery to restore the anatomy and improve symptoms in POP patients. Traditional surgical methods such as Mann's surgery, vaginal anterior/posterior wall repair, and vaginal closure surgery can all be used for POP repair surgery, but there are low success rates of anatomical reduction, no obvious symptom relief, postoperative prolapse anatomy/symptoms, etc. recurrence and other issues. As an effective surgical tool for POP repair surgery, transvaginal surgical mesh can enhance the effect of surgical repair, improve patient satisfaction, and reduce postoperative anatomy and/or symptom recurrence; however, there are surgical procedures such as exposure of mesh and increased risk of bleeding Therefore, close attention should be paid to the risk of short-term and long-term complications after surgery. Therefore, it is necessary to provide a patch made of a new material for female pelvic reconstruction.

Contents of the invention

The purpose of the present invention is to provide a warp-knitted silk fibroin patch for treating female pelvic floor dysfunction, which has excellent mechanical properties, microstructure and biocompatibility, and is suitable for female pelvic reconstruction.

The preparation method of the silk fibroin patch provided by the present invention comprises the following steps:

The silk fibroin thread is warp-knitted and then heat-set;

The diameter of the silk fibroin thread is 120-125 µm.

In the above-mentioned preparation method, the conditions of the warp knitting are as follows:

Laying yards are GB1:21/23/10/12/10/23:21//23//, GB2:00/00/33/33/00/00/33/33//, GB3:12/ 10/23:21/23/10/12/10//;

The threading method is one thread and one empty;

The let-off amounts are 2250 mm/rack, 850 mm/rack and 2250 mm/rack respectively;

RS4EL warp knitting machine can be used for weaving, and the pulling density is 10; before weaving, the silk fibroin thread can be wound on the warp beam in parallel, constant speed and neatly by using DN21 warping machine, and the number of ends can be 200 .

In the above-mentioned preparation method, the conditions of the heat setting are as follows:

The temperature is 130° C., and the time is 6 to 8 minutes.

In the silk fibroin patch prepared by the method of the invention, the silk fibroin patch has a pore diameter greater than 200 µm, a surface density of 51-53 g/m ² , and a porosity of 39-45%.

The application of the silk fibroin patch prepared by the present invention as a pelvic organ prolapse patch also belongs to the protection scope of the present invention.

The silk fibroin patch of the present invention can reduce the foreign body reaction after the patch is implanted, and has good biocompatibility; the silk fibroin patch of the present invention is softer, and it is compatible with the normal pelvic floor on the basis of meeting the mechanical support required by the pelvic floor. Tissue strength gaps are minimal, ideal for pelvic floor reconstruction patches.

Description of drawings

Figure 1 is the scanning electron micrographs of three kinds of patch, silk fibroin thread, polypropylene thread and silk thread; among them, a is the scanning electron microscope image of polypropylene patch (25X); b is the scanning electron microscope of silk fibroin patch Figure (25X); c is the scanning electron microscope image of silk fibroin polypropylene patch (25X); d is the scanning electron microscope image of polypropylene patch (100X); e is the scanning electron microscope image of silk fibroin patch (100X) ; f is the SEM image of silk fibroin polypropylene patch (100X); g is the SEM image of silk fibroin wire (1000X); h is the SEM image of silk fibroin wire (1000X); i is the SEM image of polypropylene wire ( 1000X); j is the SEM image of silk thread (5000X); k is the SEM image of silk fibroin thread (5000X).

Figure 2 is the physical performance test of polypropylene patch, silk fibroin patch and mixed patch; among them, a is the surface density comparison of the three kinds of patches; b is the thickness comparison of the three kinds of patches; c is the comparison of the three kinds of patches Porosity comparison of patches.

Figure 3 is a comparison of longitudinal and transverse breaking strength of polypropylene patch, silk fibroin patch and mixed patch.

Figure 4 is a comparison of longitudinal and transverse elongation at break of polypropylene patch, silk fibroin patch and hybrid patch.

Fig. 5 is a comparison of stiffness among polypropylene patch, silk fibroin patch and hybrid patch.

Figure 6 shows the test results of mechanical properties of polypropylene patch, silk fibroin patch and hybrid patch; among them, a is the comparison of Young's modulus of the three patches; b is the comparison of the bending stiffness of the three patches ; c is the comparison of the bursting strength of the three patches.

Figure 7 shows the tear strength test results of polypropylene patch, silk fibroin patch and mixed patch (n=5, * means p< 0.05 ).

Figure 8 shows the construction of the surgical model of partial abdominal wall defect in rats and the tissue adhesion around the patch when they were killed 12 weeks after the operation; among them, a is the model photo of partial abdominal wall muscle defect in the blank control group; b is the partial abdominal wall of the polypropylene patch group Modeling photos of muscle defects; c is the modeling photos of partial abdominal wall muscle defects in silk fibroin patch group; d is the modeling photos of partial abdominal wall muscle defects in mixed patch group; Adhesion of subcutaneous tissue; f is the adhesion of the mesh to the subcutaneous tissue in the polypropylene patch group at 12 weeks after the operation; g is the adhesion of the patch to the subcutaneous tissue in the silk fibroin patch group at the 12th week of execution; h is the Adhesion between the mesh and subcutaneous tissue in the mixed patch group at 12 weeks postoperatively (n=6).

Figure 9 shows the test results of the fracture strength of the patch tissue after four groups of rats were sacrificed at 1 week, 4 weeks and 12 weeks after operation (n=6, capital letters A and B represent the comparison between the four groups at the same treatment time point, the letters The difference between the two groups that are not the same is statistically significant (p<0.05), and the difference between the two groups with the same letter is not statistically significant (p>0.05); the lowercase letters a and b represent the comparison between different treatment time points in the same group, and the letters without letters There is a statistically significant difference between the same two treatment time points (p<0.05), but there is no statistically significant difference between the two treatment time points with the same letter (p>0.05)).

Figure 10 shows the changes in stiffness of the four groups of rats.

Figure 11 shows the HE staining results (400×) of the blank control group, polypropylene patch group, silk fibroin patch group and mixed patch group at different times (1 week, 4 weeks, 12 weeks) after operation (nucleus It is blue-purple, observe the proportion of neutrophils, and the arrows in the figure indicate foreign body giant cells (n=6)).

Figure 12 is the proportion of neutrophil counts around the patch of the blank control group, polypropylene patch group, silk fibroin patch group and mixed patch group ((n=6, capital letters A and B represent the same processing time Point four groups are compared with each other, the difference between the two groups with different letters is statistically significant (p<0.05), and the difference between the two groups with the same letter is not statistically significant (p>0.05); lowercase letters a and b indicate that the same group is different Comparing the treatment time points with each other, the difference between the two treatment time points with different letters is statistically significant (p<0.05), and the difference between the two treatment time points with the same letter is not statistically significant (p>0.05)).

Figure 13 shows the Masson staining results (100×) of the blank control group, polypropylene patch group, silk fibroin patch group and mixed patch group at different times (1 week, 4 weeks, 12 weeks) after operation (in Under the light microscope, the collagen fibers are blue, the muscle fibers, silk fibroin threads and red blood cells and other tissues are red, and the nuclei are blue-black, n=6).

Figure 14 shows the results of Sirius scarlet staining in the blank control group, polypropylene patch group, silk fibroin patch group and mixed patch group at different times (1 week, 4 weeks, 12 weeks) after operation (200×) (Type I collagen was red or bright red under a polarizing microscope, and type I collagen was green, n=6).

Figure 15 shows the staining of M2 macrophages (CD163) in the blank control group, polypropylene patch group, silk fibroin patch group and mixed patch group at different times (1 week, 4 weeks, 12 weeks) after operation Results (100×) (hematoxylin-stained cell nuclei are blue under the ordinary light microscope, and the positive expression of DAB is brownish yellow).

Figure 16 shows the macrophage counts after patch implantation in the blank control group, polypropylene patch group, silk fibroin patch group, and mixed patch group (100×) (n=6, capital letters A and B represent the same Comparing the four groups at the treatment time point, the difference between the two groups with different letters is statistically significant (p<0.05), and the difference between the two groups with the same letter is not statistically significant (p>0.05); lowercase letters a and b represent the same Compared between different treatment time points in the group, the difference between the two treatment time points with different letters is statistically significant (p<0.05), and the difference between the two treatment time points with the same letter is not statistically significant (p>0.05)).

Figure 17 shows the M-type macrophage (CD86) staining of blank control group, polypropylene patch group, silk fibroin patch group and mixed patch group at different times (1 week, 4 weeks, 12 weeks) after operation Results (100×) (hematoxylin-stained cell nuclei are blue under a common end-to-end microscope, and the positive expression of DAB is yellow).

Figure 18 shows the staining results of vascular smooth muscle actin (SMA) in the blank control group, polypropylene patch group, silk fibroin patch group and mixed patch group at different times (1 week, 4 weeks, 12 weeks) (100×) (Hematoxylin-stained cell nuclei are in yellow color under ordinary light microscope, and the positive expression of DAB is brownish yellow.

Figure 19 is the count of new blood vessels around the patch (100X) (n=6, capital letters A and B represent the comparison between the four groups at the same treatment time point, and the difference between the two groups with different letters is statistically significant (p<0.05) , the difference between the two groups with the same letter is not statistically significant (p>0.05); the lowercase letters a and b represent the comparison between different treatment time points in the same group, and the difference between the two treatment time points with different letters is statistically significant (p <0.05), there was no significant difference between the two treatment time points with the same letter (p>0.05).

Detailed ways

Embodiment 1, the preparation of patch

1) Warping: Use the DN21 warping machine to process the silk fibroin thread (Rugao Beitefu Thread Co., Ltd.) and polypropylene monofilament (Changzhou Hongxiang Medical Supplies Technology Co., Ltd.) according to the number of warp threads required by the process. And length, use parallel tension, constant speed, and neatly wound on the warp shaft for warp knitting machine use.

2) Warp knitting: use the RS4EL warp knitting machine to knit the patch according to the process parameters in the table below, and obtain a silk fibroin patch (M1) and a mixed knitting patch (M2).

3) Heat setting: Both patches are set at 130°C for 6 minutes using a 350-1000mm setting machine.

Table 1 Process parameters of warp knitted patch

Embodiment 2, the physical property of patch and microstructure

1. Experimental method

1. Electron microscope observation of patch and braided wire

1) Take the sterilized and preserved polypropylene patch (Herniamesh Co., Ltd., Italy), silk fibroin patch, mixed patch, silk thread, silk fibroin thread (Rugao Beitefu Thread Co., Ltd.), polypropylene sheet Silk (Changzhou Hongxiang Medical Supplies Technology Co., Ltd.), keep dry.

2) Conductive treatment of the patch, fix the three kinds of patches on the copper plate respectively, and spray gold for 10s. A high-performance field emission JSM-7900F scanning electron microscope was used to observe its surface structure and pore size.

2. Physical properties of patch

1) Surface density detection

The surface density is the mass of the patch per square meter. In this test, the BA110S analytical balance is used to weigh the patch sample to obtain the sample mass M (unit g). The size of the sample to be weighed is 100mm×100mm, the surface density of the sample (unit g/m ² )=M/(0.1×0.1), each sample is measured 5 times, and the average value is taken.

2) Thickness detection

Thickness is the distance between the upper and lower opposite sides of the patch. This test is measured according to the national standard GB/T3 820-1997, and YG141N digital fabric thickness gauge is used to measure the thickness of the sample. The sample size is 100mm×100mm, and the presser foot area is 2000±20mm ² . Place the sample on the reference plate, parallel to the presser foot of the plate, pressurize at 1±0.01kPa, pressurize for 30±5s and record the vertical distance between the two plates, which is the thickness of the sample (in mm) measured value. Each sample was measured 5 times in different areas, and the results were averaged.

3) Porosity detection

Porosity is the percentage of the pore area of the patch to the total area of the patch in its natural state. Use the JSM-7900F scanning electron microscope to observe the sample, choose 5 different fields of view at 25X to take pictures, import the photos into photoshop, and adjust the threshold to the optimal threshold after grayscale processing to obtain a black and white dichromatic image, through the histogram 255 The number of black pixels in the pore area Nb and the number of total pixels Nt are obtained by the color scale processing, and the calculated porosity (unit %)=Nb/Nt×100%. Each sample is selected for 5 calculations in different areas, and the results are averaged.

4) Fracture strength test

Breaking strength is the maximum force at which the patch is stretched under specified conditions until it breaks. AGS-X electronic universal testing machine is selected to test the tensile properties of the test patch. The test conditions refer to the national standard GB/T 3923.1-1997. The pulling speed of the testing machine is set to 100mm/min, and the clamping distance is 200mm, the pre-tension is 1N. The breaking strength is the maximum force (in N) recorded when the test sample is broken during the tensile test. According to the longitudinal and transverse direction of the sample, cut the test samples with the specification of 50mm×300mm respectively, test 5 times each, and take the average value of the results.

5) Detection of elongation at break

Elongation at break is the ratio of the elongation of the patch at the maximum force during stretching to the length of the patch before stretching. AGS-X electronic universal testing machine was selected to test the tensile properties of the test patch. The pulling speed of the testing machine was set at 100mm/min, the clamping distance was L (200mm), and the pre-tension was 1N. The increment △L (in mm) of the sample length corresponding to the breaking strength during the stretching of the sample to the breaking process, and the elongation at break (in %)=△L/L×100%. According to the longitudinal and transverse direction of the sample, cut the test samples with the specification of 50mm×300mm respectively, test 5 times each, and take the average value of the results.

6) Stiffness detection

Stiffness is the ability of the patch to resist elastic deformation when a force is applied. The experimental method is the same as 5), the force F (unit N) acting on the patch during the test sample stretching to fracture, the deformation due to the force is the sample elongation increment △L (unit cm), and the stiffness K (unit N/cm) = F/△L, the test sample size is 50mm×10mm, cut the sample according to the vertical and horizontal directions of the sample for 5 times, and take the average value of the results.

7) Young's modulus detection

Young's modulus is a physical quantity that describes the ability of a patch to resist deformation. The experimental method is the same as 6), A (unit mm ² ) is the cross-sectional area of the test sample, L (unit mm) is the length of the sample, that is, the clamping distance, and the stress F (unit N) of the test sample during stretching to fracture , the sample elongation increment △L (unit mm) corresponding to the stress F, Young's modulus E (N/mm ² )=(F•L)/(A•△L). The size of the test sample is 50mm×300mm, the test is performed 5 times, and the results are averaged.

8) Bending stiffness detection

Bending stiffness is the ability of the patch to resist its bending deformation, reflecting the stiffness of the patch. According to the national standard GB/T 18318-2001, place a rectangular sample with a size of (25±1mm×(250±1)mm on a horizontal platform, and the long axis of the sample is parallel to the long axis of the platform. Propel the sample in the direction of the long axis so that it protrudes from the platform and bends under its own weight. The end of the protruding part is suspended in the air, and the other end of the sample that is still on the platform is pressed by a ruler. When the head end of the sample passes through the front edge of the platform and reaches On a slope with an inclination angle of 41.50 to the horizontal line, the protruding length is equal to twice the bending length of the sample, from which the bending length can be calculated. Therefore, half of the protruding length is taken as the bending length, and four bending lengths are recorded for each sample. From this, the average bending length of each sample was calculated.

Bending stiffness G (unit mN.cm) = m×C ³ ×10 ^-2 , where m is the mass per unit area of the sample, unit g/m ² ; C is the average bending length of the sample, unit cm. The test was performed 5 times, and the results were averaged.

9) Bursting strength test

Bursting strength is an indicator of durability when the patch is deformed or ruptured by external force. In this test, according to the national standard GB/T19976-2005, the AGS-X electronic universal testing machine is selected for testing. The spherical mandrel pushes vertically towards the sample at a constant speed of 300mm/min±10mm/min, and the maximum force measured during the deformation of the sample until it breaks is the bursting strength (in N). The inner diameter of the ring holder is 45mm±0.5mm, the head end of the ejector rod is a polished steel ball, and the diameter of the ball is 25mm±0.02mm. The test was performed 5 times, and the results were averaged.

10) Tear strength test

Tear strength is the ability of the patch to resist tearing failure. This test is carried out according to the national standard GB/T3917.2-2009, the sample size is (200±2)mm×(50±1)mm, the sample is cut from the middle of the width direction to a length of (100±1) mm parallel to the longitudinal direction of the split, the tear end point is marked at (25 ± 1) mm from the uncut end of the sample. Clamp each of the two trouser legs of the sample into a clamp, the distance between the two clamps is 100mm, the cutting line is aligned with the center line of the clamp, and the sample is continuously torn to the end of the sample at a tensile speed of 100mm/min mark. Record the maximum tear strength (N). According to the vertical and horizontal shear samples of the sample test 5 times, the results are averaged.

2. Experimental results

1. Electron microscope observation results of patches and braided wires

After spraying gold, observe the structures and surface conditions of the three patches under a scanning electron microscope with a magnification of 25 times and a lens of 100 times. It can be observed that the polypropylene threads used in the braided polypropylene patch and the mixed patch are all single The single-strand silk fibroin thread used in weaving silk fibroin patches and hybrid patches is made of dozens of silk fibroin monofilament condensed strands.

The mesh diameter of the polypropylene patch mainly fluctuates between 470 μm and 1480 μm (a in Figure 1), and there are some pores smaller than 300 μm between the polypropylene braided wires. The mesh diameter of the silk fibroin patch is between 270µm and 1680µm (b in Figure 1), and the large pores are mainly larger than 500µm. The mesh diameter of the hybrid patch is between 220µm and 1100µm (c in Figure 1), mainly with a diameter above 500µm, and there are some pores with a diameter smaller than 100µm between the braided wires.

The diameter of the single polypropylene wire of the braided polypropylene patch is about 90 µm (d in Figure 1), while the diameter of the polypropylene single wire used in the hybrid patch is about 125 µm (f in Figure 1), and the surface of the polypropylene single wire used is smooth and burr-free (i in Figure 1).

The surface of silk fibroin thread before degumming is covered with a layer of sericin (g in Figure 1, j in Figure 1), and the silk fibroin monofilament wrapped in it is revealed after degumming (h in Figure 1, k in Figure 1) . The diameter of the silk fibroin threads used in weaving silk fibroin patches and mixed patches is about 200 μm, and there are rough burr-like structures on the surface of some silk fibroin threads after silk desericinization (Fig. 1 e, Fig. 1 middle f, h) in Figure 1, the warp knitting process makes the structure of the silk fibroin patch and the mixed patch more complex and three-dimensional.

2. Analysis of the physical and mechanical properties of the patch

1) Surface density

Areal density is the mass of the patch per square meter. Comparison of areal densities of the three patches (Table 2, a in Figure 2): polypropylene patch < silk fibroin patch < mixed patch, the difference was statistically significant ( p<0.05 ).

2) Thickness

The thickness comparison of the three kinds of patches (Table 2, b in Figure 2): Polypropylene patch<silk fibroin patch<mixed patch, the difference was statistically significant ( p<0.05 ).

3) Porosity

Comparing the porosity of the three kinds of patches (Table 2, c in Figure 2), the porosity of the polypropylene patch is greater than that of the silk fibroin patch and the mixed patch, and the difference is statistically significant ( p<0.05 ). The porosity of the silk fibroin patch and the mixed patch were both lower than 40%, and there was no statistically significant difference between them ( p=0.7284 ). Table 2 shows the physical properties of the three patches (x±SD).

Table 2 Physical property testing of polypropylene patch, silk fibroin patch and mixed patch

4) Breaking strength

The longitudinal and transverse breaking strength measurements of the three kinds of patches are as follows (Table 3, Figure 3). The longitudinal breaking strengths of the three kinds of patches are all >300N, among which the mixed patch has the highest breaking strength, and the silk fibroin patch breaks The strength is the smallest, but the difference between the three patches is not statistically significant ( p>0.05 ); the transverse breaking strength of the polypropylene patch is significantly greater than that of the silk fibroin patch and the mixed patch, and the difference is statistically significant ( p< 0.05 ), the transverse breaking strength of the silk fibroin patch was slightly greater than that of the mixed patch, and the difference was not statistically significant ( p=0.3255 ). There were statistically significant differences between the longitudinal breaking strength and the transverse breaking strength of the three patches ( p<0.05 ).

5) Elongation at break

The longitudinal and transverse elongation at break of the three kinds of patches (Table 3, Figure 4) were all polypropylene patches < hybrid patches < silk fibroin patches, and the differences were statistically significant ( p<0.05 ). There was a statistically significant difference between the longitudinal breaking elongation of the silk fibroin patch and its own transverse breaking elongation ( p<0.05 ). There was no significant difference in the longitudinal and transverse elongation at break between the polypropylene patch and the hybrid patch ( p>0.05 ).

Table 3 Mechanical properties of the three patches (x±SD)

6) Rigidity

Stiffness is the ability of the patch to resist elastic deformation when it is stressed, and the greater the stiffness, the harder it is for the patch to undergo elastic deformation when it is stressed. The longitudinal stiffness of the three kinds of patches was compared (Table 4, Figure 5): polypropylene patch > mixed patch > silk fibroin patch, and the difference was statistically significant ( p<0.05 ). The transverse stiffness of the polypropylene patch was also significantly greater than that of the silk fibroin patch and the mixed patch, and the difference was statistically significant ( p<0.05 ), but there was no statistically significant difference in the transverse stiffness between the silk fibroin patch and the mixed patch ( p = 0.2775 ). There were statistically significant differences between the longitudinal breaking strength and the transverse breaking strength of the three patches ( p<0.05 ).

Table 4 Longitudinal and transverse stiffness of the three patches (x±SD)

7) Young's modulus

Both Young's modulus and stiffness are physical quantities that describe the ability of a patch to resist deformation. Comparison of Young's modulus of the three kinds of patches (Table 5, a in Figure 6): Polypropylene patch > Mixed patch > Silk fibroin patch, the difference was statistically significant ( p<0.05 ).

8) Bending stiffness

Bending stiffness is the ability of the patch to resist its bending deformation, reflecting the stiffness of the patch. The greater the bending stiffness, the stiffer the patch; the lower the bending stiffness, the softer the patch. The bending stiffness of the polypropylene patch and the mixed patch were significantly greater than that of the silk fibroin patch (Table 5, b in Figure 6), and the difference was statistically significant ( p<0.05 ). There was no significant difference in the bending stiffness between the polypropylene patch and the hybrid patch ( p>0.05 ).

9) Breaking strength

The bursting strength reflects the durability index of the patch when it is deformed or ruptured by external force. The bursting strengths of the three patches were all greater than 350N (Table 5, c in Figure 6), and there was no statistically significant difference among them (p>0.05).

Table 5 Mechanical properties of the three patches (x±SD)

10) Tear strength

Tear strength is the ability of the patch to resist tearing failure. The longitudinal and transverse tearing strength comparisons of the three kinds of patches (Table 6, Figure 7): polypropylene patch > silk fibroin patch > mixed patch, but the difference in longitudinal tearing strength among the three kinds is not statistically significant Significant ( p>0.05 ). The transverse tear strength of the hybrid patch was significantly lower than that of the polypropylene patch and the silk fibroin patch, and the difference was statistically significant ( p<0.05 ). The transverse tear strength of the polypropylene patch was slightly greater than that of the silk fibroin patch, and the difference was not statistically significant ( p=0.0548 ). There is a statistically significant difference between the longitudinal tear strength and the transverse tear strength of the mixed patch itself ( p<0.05 ). There was no significant difference in longitudinal tear strength and transverse tear strength between polypropylene patch and silk fibroin patch ( p>0.05 ).

Table 6 Longitudinal and transverse tearing strength of 3 patches (x±SD)

3. Comparison of experimental results

1. Comparison of the physical properties of the three patches

Infection after mesh implantation may lead to mesh exposure or erosion. An ideal POP surgical mesh needs to have biomechanical response, biocompatibility, and anti-infection properties suitable for pelvic floor tissue. Several studies have shown that the lower the surface density of the patch and the larger the pore size, the lower the chance of infection, the less obvious the foreign body reaction is observed, and the less bridging of scar. However, if the areal density is too low, there are also problems such as patch folding and surgical operation difficulties. The selection of the patch areal density needs to explore a suitable range to ensure optimal mechanical properties and biocompatibility.

In addition to the mesh pore diameter, the gap between the individual filaments of the mesh is also an important factor affecting postoperative complications. Inflammatory and fibrous responses were more pronounced and persistent after implantation of multifilament mesh in the abdominal wall of rats compared with monofilament mesh. If the pores are small (<10µm), leukocytes (9~15µm) and macrophages (16~20µm) are not allowed to pass through and cannot participate in the immune response against infection, but bacteria (<2µm) can perfectly colonize the patch hole, increasing the risk of infection. In addition, mesh diameters <10 µm or multifilament materials may lead to postoperative infection; while mesh diameters >100 µm not only facilitate the migration of macrophages and leukocytes, reduce the risk of infection and mesh-related complications, but also allow adult Adhesive engraftment of fibroblasts produces more collagen fibers, allowing for better incorporation of the patch into the host tissue. The pore size of the patch not only affects the migration of bacteria and inflammatory cells, the growth of new tissue, but also affects the flexibility of the patch. Meshes with smaller pore sizes are stiffer, increasing the risk of mesh erosion, postoperative pelvic pain, and functional impairment. Large-pore, monofilament, lightweight mesh is relatively more suitable for POP repair surgery.

The research results of the present invention show that the areal density of the silk fibroin patch is slightly greater than that of the polypropylene patch, but both are less than 60g/m ² , both of which belong to the middle-weight patch; the areal density of the mixed patch is More than 70g/m ² , it belongs to heavy patch. The three-dimensional structure of the silk fibroin patch and the hybrid patch is more complex than that of the polypropylene patch, which is speculated to be more suitable for cell adhesion and migration, and conducive to the growth of new tissue.

In addition, most of the pore diameters of the three kinds of patches are larger than 200 μm, allowing the migration of inflammatory cells and the growth of new tissues, but the silk fibroin threads used in the silk fibroin patch and the mixed patch are There are some small pores less than 100 μm in between, and there is a risk of infection after the patch is implanted in the body. Further in vivo experiments are needed to study the biocompatibility of the patch.

2. Comparison of the mechanical properties of the three patches

Increased pelvic-abdominal pressure and weakening of the supporting muscles and fascia of the abdominal wall and pelvic floor are the main causes of abdominal wall hernia and POP. The intra-abdominal pressure is about 4mmHg in the resting supine state, and it is 5-6 times that in the supine state in the resting upright state. When there are occasional high-pressure events in the abdomen caused by coughing, etc., the maximum measured value of intra-abdominal pressure can reach 180mmHg. The stress on the abdominal wall muscle and repair mesh can reach 16N/cm, while the pressure on the mesh after abdominal wall hernia repair is 2N/cm in resting state.

After patch repair surgery in POP patients, pelvic-abdominal pressure will also act on the pelvic floor support tissue and the surgical patch. Data on the mechanical properties required for the use of mesh for POP repair are lacking, and it is generally accepted that the mechanical properties of pelvic floor mesh are greater than those required for abdominal defect repair.

However, if the stiffness of the implanted mesh is too high, the tissues around the implanted site may undergo disuse atrophy under the stress barrier of the mesh, and the mesh will be exposed and eroded. Feola et al. implanted meshes with different rigidities into macaques for sacral colpopexy after hysterectomy, and found that the mechanical properties of macaque vaginal tissue deteriorated the most after the mesh with the highest stiffness was implanted, and the vaginal contractility and vaginal tissue stiffness decreased the most. obvious. The in vivo experiment of Liang et al. in macaques also supports this conclusion. The experiment found that the implantation of high-rigidity and hard mesh significantly thinned the vaginal smooth muscle layer, increased cell apoptosis around the mesh, and decreased the content of collagen and elastin. It is due to the effect of stress shielding.

The research results of the present invention show that the silk fibroin patch and the silk fibroin polypropylene mixed patch prepared by the present invention have similar longitudinal breaking strength and bursting strength to the clinically used polypropylene patch. However, the stiffness and bending stiffness of the silk fibroin patch are lower, and the texture of the patch is softer. On the basis of meeting the mechanical support required by the pelvic floor, the gap between the strength of the silk fibroin patch and the normal pelvic floor tissue is the smallest. The mechanical properties such as stiffness and bending stiffness of the hybrid patch are basically the same as those of the polypropylene patch, and they are significantly different from the mechanical properties required by normal pelvic floor tissue. It is speculated that after the silk fibroin patch is implanted in the body, it is better integrated with the surrounding tissue, and the formed patch-tissue complex is more suitable for the support of the pelvic floor.

From the above analysis, it can be seen that the microstructure of the silk fibroin patch and the hybrid patch shows that the surface of the silk thread is rough, and the multifilament weave has a more complex three-dimensional structure than the polypropylene patch. Adhesion and migration, facilitating the growth of inflammatory cells and new tissues. The mechanical properties such as breaking strength and bursting strength of the silk fibroin patch can meet the mechanical requirements of the clinical use of the polypropylene patch; the mixed patch still has similar bending stiffness and texture as the polypropylene patch, and the texture is more rigid. The flexural stiffness of the silk fibroin patch is significantly reduced, and the texture is softer under the premise of meeting the rigidity required by the pelvic floor pressure. It is an ideal choice for pelvic floor reconstruction patches.

Embodiment 3, the biocompatibility research of patch

1. Experimental method

(1) patch preparation

1) Make the 3 kinds of patches separately into bags of 2×2cm size;

2) Use ethylene oxide to sterilize the subpackaged patches, and store them in a fume hood for more than two weeks for later use.

(2) Animal experiments

1) Grouping

120 SD female rats were divided into 4 groups, 30 in the polypropylene patch group, 30 in the silk fibroin patch group, 30 in the mixed patch group, and 30 in the blank control group. All underwent partial abdominal wall muscle defect surgery.

) Construction of a partial abdominal wall muscle defect model in rats and defect repair surgery

Rats were anesthetized by isoflurane inhalation at 5cc/min, and maintained at a dose of 2cc/min after the anesthesia was satisfactory. The limbs were fixed, the abdominal hair was shaved off with a shaver, and the abdominal skin was disinfected three times with 75% alcohol. A 3 cm longitudinal incision was made on the lower abdomen, and the skin and subcutaneous tissue were bluntly separated. The abdominal wall muscle layer was cut longitudinally at 2 cm on the left and right sides of the linea alba, and the internal oblique muscle and transverse abdominis were bluntly separated. A partial abdominal wall muscle defect with a size of 1×1cm, with the transversus abdominis and peritoneum retained.

In the experimental group, a 2×2 cm patch was covered on the defect, and 5-0 absorbable surgical sutures were used to fix the edge of the patch to the surrounding tissue of the abdominal wall defect without tension. Position four-point stitching. The incision was sutured layer by layer and disinfected with 75% alcohol. The blood loss was 1~2ml during the operation, and the pain relief jelly was used for comfort after the operation.

3) Take materials

The rats were fed routinely after the operation, and sacrificed at 1 week, 4 weeks, 12 weeks, half a year and 1 year after the operation, and 6 rats were sacrificed in each group each time. After the rats were killed, the left and right sides of the experimental group with implanted patches and some surrounding tissues and the abdominal wall tissues with surgical sutures in the control group were taken out. The mechanical properties of the same rat were tested immediately after taking out one side of the tissue, and the other side of the tissue was soaked. Fix in 4% paraformaldehyde for 24 hours.

The adhesion between the surgical site and the subcutaneous tissue was scored according to the Walker scoring standard when collecting materials: 0 points: no adhesion; 1 point: adhesion area < 25%, which can be bluntly separated by small force; 2 points: adhesion area ≥ 25% %, can be bluntly separated by a large force; 3 points: tight adhesion, sharp separation is required.

(3) Mechanics performance test of patch tissue

AGS-X electronic universal testing machine is selected to test the tensile properties of the test tissue. Due to the limited size of the tissue, the test sample size is 20mm×20mm. The test conditions are adjusted according to the standard GB/T 3923.1-1997. The testing machine The pulling speed is set to 10mm/min, the clamping distance is 10mm, and the pre-tension is 1N. During the tensile test of the test sample, the maximum force recorded when the sample is broken is the breaking strength (unit N). Breaking strength is an absolute value representing the strength of a material.

Stiffness refers to the ability of a material or structure to resist elastic deformation when it is stressed, and it is a characterization of the difficulty of elastic deformation of a material or structure. Record the force F (unit N) acting on the patch during the stretching of the test sample to fracture, the growth of the sample due to the force, that is, the deformation amount △L (unit cm), and calculate the sample stiffness K (unit N/ cm) Tensile force F (in N).

(4) Tissue embedding and fixation

1) Obtaining materials: After the fresh tissue was fixed with 4% paraformaldehyde for more than 24 hours, the tissue block was taken out in the fume hood and the target tissue was trimmed with a scalpel, and the trimmed tissue was placed in the marked dehydration box.

2) Dehydration: 5% alcohol 4h→85% alcohol 2h→90% alcohol 2h→95% alcohol 1h→absolute ethanol I30min→absolute ethanol II30min→alcohol benzene 5~10min→xylene I5~10min→xylene II5- 10min→paraffin I1h→paraffin II1h→paraffin III1h.

3) Embedding: Place the wax-soaked tissue in the embedding machine for embedding, and cool it on a -20° freezing table. After the wax solidifies, take out the wax block from the embedding frame and trim the wax block.

4) Slicing: Place the trimmed wax block on a paraffin microtome and slice it with a thickness of 3-4µm. The slices were floated on the 40°C warm water of the slide machine to flatten the tissue, pasted on the anti-off slides and numbered, and put into a 60°C oven to bake the slices. After the water is dried and the wax is roasted, take it out and store it at room temperature for later use.

(5) HE staining

Randomly select two slices from each tissue wax block for HE staining, specific steps:

1) Bake slices: Bake slices in a 65-degree oven for 1 hour.

2) Dewaxing to water: put the slices in xylene Ⅰ for 20 minutes→xylene Ⅱ for 20 minutes→absolute ethanol Ⅰ for 10 minutes→absolute ethanol Ⅱ for 10 minutes→95% alcohol for 5 minutes→90% alcohol for 5 minutes→80% alcohol for 5 minutes→70% alcohol for 5 minutes→ Wash with distilled water for 1 min.

3) Hematoxylin staining of 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, turn blue with 0.6% ammonia water, and rinse with running water.

4) Stain the cytoplasm with eosin: Put the sections in eosin staining solution for 1-3 minutes, wash with tap water for 1 minute.

5) Dehydration and sealing: put the slices in 95% alcohol I5min→95% alcohol II5min→absolute ethanol I5min→absolute ethanol II5min→xylene I5min→xylene II5min for dehydration and transparency, take the slices out of xylene for a while After drying, the slides were sealed with neutral gum.

6) Interpretation: Judgment of results under a microscope, image collection and analysis.

Staining results: the nuclei were blue-purple.

7) Inflammatory cell count

Inflammatory cell counting: HE slices were placed under a 400× optical microscope, and 5 fields of view were counted for each slice, and the proportion of neutrophils in each field of view was calculated and compared, and the results were averaged.

6) Masson staining

Masson staining is one of the main methods to display fibers in tissues, and it is mainly used to identify collagen fibers and muscle fibers. In order to identify the components of the new tissue around the patch, two sections of tissue wax blocks at 1 week, 4 weeks, and 12 weeks after surgery were randomly selected for Masson staining. For specific steps, refer to the kit instructions.

1) Bake slices: Bake slices in a 65-degree oven for 1 hour.

2) Dewaxing to water: put the slices in xylene Ⅰ for 20 minutes→xylene Ⅱ for 20 minutes→absolute ethanol Ⅰ for 10 minutes→absolute ethanol Ⅱ for 10 minutes→95% alcohol for 5 minutes→90% alcohol for 5 minutes→80% alcohol for 5 minutes→70% alcohol for 5 minutes→ Wash with distilled water for 1 min.

3) Hematoxylin staining of nuclei: Stain with Weigert’s iron hematoxylin in the Masson staining kit for 5 minutes, wash in tap water, differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse in tap water for 1-3 minutes, turn blue with 1% ammonia water for 30 seconds, wash in tap water for 1-3 minutes.

4) Ponceau staining: Ponceau red acid fuchsin solution in the Masson staining kit was used for 5-10 minutes, and rinsed quickly with distilled water.

5) Phosphomolybdic acid treatment: add dropwise the aqueous solution of phosphomolybdic acid in the Masson staining kit, and stain for about 3 to 5 minutes.

6) Aniline blue staining: counterstain with the aniline blue solution in the Masson staining kit for 5 minutes, and differentiate with 1% glacial acetic acid solution for about 1 minute.

7) Dehydration and sealing: put the slices in 95% alcohol I5min→95% alcohol II5min→absolute ethanol I5min→absolute ethanol II5min→xylene I5min→xylene II5min for dehydration and transparency, take the slices out of xylene for a while After drying, the slides were sealed with neutral gum.

8) Interpretation: Judgment of results under a microscope, image collection and analysis.

Staining results: collagen fibers are blue; other tissues such as muscle fibers, cellulose, and red blood cells are red; nuclei are blue-black.

7) Sirius Scarlet Dyeing

1) Bake slices: Bake slices in a 65-degree oven for 1 hour.

2) Dewaxing to water: put the slices in xylene Ⅰ for 20 minutes→xylene Ⅱ for 20 minutes→absolute ethanol Ⅰ for 10 minutes→absolute ethanol Ⅱ for 10 minutes→95% alcohol for 5 minutes→90% alcohol for 5 minutes→80% alcohol for 5 minutes→70% alcohol for 5 minutes→ Wash with distilled water for 1 min.

3) Staining with Sirius Scarlet staining solution: Place the slices in saturated picric acid Sirius Scarlet staining solution for 8 minutes.

4) Rinsing: Rinse the slices in anhydrous alcohol for several minutes until the observation under the microscope is satisfactory.

5) Seal the slices: After drying the slices in a 60°C oven, place them in xylene for 5 minutes and seal them with neutral gum.

6) Interpretation: Polarizing microscope inspection, image acquisition and analysis.

Staining results: type I collagen is red or bright red; type II collagen is orange-yellow, and type III collagen is green. Five different fields of view were selected for each slice to take pictures, and Image-Pro Plus 6.0 was used to measure the area of type I collagen and type III collagen in the picture, and calculate the collagen volume ratio.

(8) Immunohistochemical staining

1) Bake slices: Bake slices in a 65-degree oven for 1 hour.

2) Dewaxing to water: Put the slices in xylene for 7 min→xylene II for 7 min→xylene II for 7 min→absolute ethanol for 20 s→95% ethanol for 20 s→75% ethanol for 20 s→distilled water for 1 min.

3) Antigen retrieval: Select the appropriate method according to the antigen to be detected, refer to the instruction manual of the primary antibody, pH 6.0 citrate antigen retrieval solution or pH 8.0 EDTA antigen retrieval solution, bathe in 95°C water for 15-20 minutes; slice after natural cooling Shake and wash in PBS 3 times, 3 min each time.

4) Block endogenous peroxidase: Add 3% H ₂ O ₂ dropwise, incubate at room temperature in the dark for 10 minutes; place the slices in PBS and shake and wash 3 times, 3 minutes each time.

5) Serum blocking: Shake the slices to dry, add 10% goat serum blocking solution dropwise to evenly cover the tissue, and seal at room temperature for 1 hour.

6) Add primary antibody: Gently suck up the blocking solution, drop the primary antibody prepared in PBS in a certain proportion on the slice, place the slice flat in a humid box and incubate overnight at 4°C.

7) Rewarming: The next day, take out the wet box, rewarm at room temperature for 30 minutes, then rinse with PBS 3 times, 3 minutes each time.

8) Add secondary antibody: after drying the slices slightly, add a secondary antibody corresponding to the species of the primary antibody (HRP-labeled goat anti-mouse/rabbit IgG polymer) to cover the tissue, incubate at room temperature for 30 minutes; wash the slices in PBS for 3 times, 3 minutes each time.

9) DAB color development: freshly prepared DAB color development solution (A liquid: B liquid = 1:20) color development, control the color development under a microscope at room temperature for 1~5min, the positive color is brownish yellow, tap water to stop the color development in time.

10) Counterstain cell nuclei: counterstain cell nuclei with Harris hematoxylin for 1-1.5 minutes, wash in tap water for 1 minute, differentiate with 1% hydrochloric acid alcohol for 3-5 seconds, rinse in tap water for 1 minute, turn blue with ammonia water for 5-10 seconds, and rinse in running water for 1 minute.

11) Dehydration and sealing: slices are placed in 95% ethanol I10s-95% ethanol II10s~95% ethanol III10s-absolute ethanol I10s-absolute ethanol II10s-absolute ethanol III10s-xylene I1min-xylene II1min for dehydration and transparency , neutral gum mount.

12) Interpretation: Judgment of results under a microscope, image collection and analysis.

Staining results: hematoxylin-stained nuclei are blue, and the positive expression of DAB is brownish yellow.

2. Statistical analysis

The above data were entered into the GraphPad Prism Version 8.2.1 software, and the data variables were normalized by the Kolmogorov-Smironov test. The normal distribution was expressed as mean ± standard deviation (x ± SD). One-way analysis of variance was used for comparison between groups, and p<0.05 was considered to have a significant statistical difference.

2. Experimental results

1. Construction of animal models

SD female rats were divided into 4 groups, all of which underwent partial abdominal wall muscle defect surgery, namely: blank control group, polypropylene patch group, silk fibroin patch group, and mixed patch patch group. The partial defect model of abdominal wall muscle in rats was successfully constructed (a in Fig. 8, b in Fig. 8, c in Fig. 8, d in Fig. 8), sutured by simple surgical suture, put in polypropylene patch, and silk fibroin patch respectively. slices and mixed patches. After routine feeding, all operated animals survived, and no problems such as animal infection, patch exposure, and patch erosion were found.

In the blank control group, only a small amount of adhesions were formed at the surgical site at 1 week and 4 weeks after surgery, and the adhesion area was less than 25%. At 12 weeks after surgery (Fig. 8 e), most of the surgical site was covered by fibrous connective tissue, but Can be bluntly separated.

In the polypropylene patch group, there was only a small amount of connective tissue on and around the material at 1 week after surgery, and the adhesion area was less than 25%, which could be bluntly dissected. The fiber wrapping increased at 4 weeks after surgery, and at 12 weeks after surgery (Fig. 8 f). A large amount of fibrous connective tissue is covered on and around the patch, closely adhered to the subcutaneous tissue, and sharp separation is required.

In the silk fibroin patch group, at 1 week and 4 weeks after surgery, a moderate amount of fibrous connective tissue was covered on and around the patch, 50%> adhesion area> 25%, and blunt separation was possible; 12 weeks after surgery (Figure 8 In g), the fiber package is more than before, but it can still be bluntly separated.

In the mixed mesh group, there were multiple layers of fibrous connective tissue above and around the mesh at 1 and 4 weeks after operation, and the adhesion area was >50% at 4 weeks after operation, but blunt separation was possible; at 12 weeks after operation (Fig. In h) of 8, a large amount of fibrous connective tissue is wrapped, and part of it is closely adhered to the subcutaneous tissue, and sharp separation is required.

2. Rat tissue force measurement results

1) Breaking strength

1) Four groups of breaking strength changes with time

1. The tissue force in the operation area of the blank control group was about 10N at 1 week, 4 weeks and 12 weeks after operation, and there was no statistically significant difference between them (p>0.05).

2. In the polypropylene patch group, there was a statistically significant difference in the increase in the breaking strength of the patch tissue in the surgical area at 4 weeks after surgery compared with 1 week after surgery ( p< 0.05); at 12 weeks after surgery, the increase in breaking strength tended to be Stable, and there was no statistically significant difference in growth compared with 4 weeks after operation ( p>0.05 ).

3. The breaking strength of the silk fibroin patch group reached the highest peak at 1 week after operation, and the breaking strength decreased significantly at 4 weeks after operation, and the difference was statistically significant ( p<0.05 ); 12 weeks after operation was compared with 4 weeks after operation The breaking strength increased slightly, and the difference was not statistically significant ( p>0.05 ).

4. The breaking strength of the mixed patch group increased rapidly, and there was no significant difference in the breaking strength at 1 week, 4 weeks and 12 weeks after surgery (p>0.05).

2) Differences in the breaking strength of the four groups at each treatment time point (Table 7, Figure 9)

1. One week after operation, the breaking strength of the blank control group was significantly lower than that of the other three patch groups, and the difference was statistically significant (p<0.05); there was no statistically significant difference among the three patch groups (p> 0.05).

2. At 4 weeks and 12 weeks after surgery, the breaking strength of the blank control group and the silk fibroin patch group was significantly lower than that of the polypropylene patch group and the mixed patch group, and the difference was statistically significant (p<0.05) .

Table 7 Comparison of the breaking strength of patch tissue in the four groups of rats at 1 week, 4 weeks, and 12 weeks after operation (x±SD)

(2) Stiffness

1) Four groups of stiffness changes with time

1. The stiffness measurement results of the surgical area tissue in the blank control group increased slightly over time, but there was no statistically significant difference between the three treatment time points ( p>0.05 ).

2. In the polypropylene mesh group, there was a statistically significant increase in the stiffness of the mesh tissue in the surgical area at 4 weeks after surgery compared with 1 week after surgery ( p<0.05 ), and it tended to be stable at 12 weeks after surgery, which was significantly higher than that at 4 weeks after surgery. There was no significant difference in weekly growth (p>0.05).

3. The stiffness of the silk fibroin patch group reached its peak at 1 week after surgery, and decreased significantly at 4 weeks after surgery, with a statistically significant difference (p<0.05). The stiffness at 12 weeks after surgery was slightly lower than that at 4 weeks after surgery. Growth, the difference was not statistically significant (p>0.05).

4. The stiffness of the mixed patch group increased rapidly, with a slight increase at 4 weeks after surgery compared with 1 week after surgery, and a slight decrease in stiffness at 12 weeks after surgery compared with 4 weeks after surgery, but there was no difference between the three treatment time points. Statistically significant ( p>0.05 ).

2) Differences in stiffness of the four groups at each treatment time point (Table 8 and Figure 10)

1. One week after operation, the breaking strength of the blank control group was significantly lower than that of the other three patch groups, and the difference was statistically significant ( p <0.05 ).

2. At 4 weeks and 12 weeks after surgery, the breaking strength of the blank control group and the silk fibroin patch group was significantly lower than that of the polypropylene patch group and the mixed patch group, and the difference was statistically significant ( p<0.05 ) .

3. The stiffness of the polypropylene mesh group and the mixed mesh group were basically the same at 1 week after operation, and the difference gradually increased at the last 4 weeks and 12 weeks after operation, polypropylene mesh group>mixed mesh group, but there was no statistically significant difference between them ( p >0.05 ).

Table 8 Comparison of patch tissue stiffness of four groups of rats at 1 week, 4 weeks and 12 weeks after operation (x±SD)

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

(1) Analysis of HE staining results

1. In the blank control group, a small amount of inflammatory cells, mainly lymphocytes, could be seen at 1 week after operation, and the inflammatory cells gradually decreased at 4 and 12 weeks after operation, and only a small amount of loose connective tissue could be seen (Figure 11 ).

2. In the polypropylene patch group, a large number of inflammatory cells can be seen on the surface of the polypropylene monofilament at 1 week after operation, mainly neutrophils. At 4 and 12 weeks after operation, the neutrophil count decreased significantly, and a small amount of foreign body giant cells could be seen, and there was more connective tissue around the patch than at 1 week after operation (Figure 11).

3. In the silk fibroin patch group, more neutrophils can be seen around the material at 1 week after operation, and there are more foreign body giant cells than the poly-patch group (Figure 11). More connective tissue surrounded; 4 weeks and 12 weeks after operation, the connective tissue gradually grew into the silk fibroin patch and wrapped the silk fibroin monofilaments, and the arrangement was regular. At 12 weeks after the operation, the widening of the silk fibroin monofilament gap and the surrounding connective tissue can be observed, indicating the degradation of silk fibroin in vivo (Figure 11).

4. The mixed patch group can count the most inflammatory cells around the silk fibroin and polypropylene threads at 1 week after operation, mainly neutrophils, which are significantly more than the polypropylene patch group and silk fibroin. Protein patch group ( p<0.05 ). At 4 and 12 weeks after surgery, the neutrophils decreased significantly, and the inflammatory cells were mainly monocytes and lymphocytes, and there were a large number of foreign body giant cells, and the connective tissue gradually wrapped and grew into the patch (Figure 11) , the statistical analysis of the neutrophils around the patch in each group is shown in Figure 12.

2) Analysis of Masson staining results

The results of Masson staining (Figure 13) showed that the new tissues in each group were mainly collagen fibers, and as time went by, more collagen fibers were formed and the structure was more compact. At 12 weeks after operation, the new collagen fibers in the polypropylene patch group could tightly surround the patch and be arranged in an orderly manner; the new collagen fibers in the silk fibroin patch group and the mixed patch group could grow into the patch and wrap the silk Protein threads form tight junctions.

3) Analysis of Sirius Scarlet staining results

The distribution and type of new collagen fibers in the surgical area or around the patch in the four groups at different treatment time points (Figure 14), and the proportions of type I collagen and type III collagen in each group at different time points are as follows (Table 9) , the results showed that the new fibers in the blank control group and the polypropylene patch group were mainly composed of type III collagen at 1 week, 4 weeks and 12 weeks after operation, and only a small amount of type Ⅰ collagen could be seen; the silk fibroin patch At 12 weeks postoperatively, more type Ⅰ collagen was induced by the mixed patch, and new collagen fibers grew into and wrapped around the surgical patch, arranged in an orderly manner and closely connected.

Table 9 The proportion of type Ⅰ and type Ⅲ collagen in different implantation time and each group

4) Analysis of immunohistochemical staining results

Immunohistochemical staining showed that M2 macrophages were predominant in the surgical site or around the patch in the silk fibroin patch group and the mixed patch group (Table 10, Figure 15 and Figure 16). Phagocytes mainly existed at the contact surface between the implanted mesh, subcutaneous tissue and abdominal wall muscles, and the number of M2 macrophages in the mixed mesh group was the highest (p<0.05) in the gap between the braided mesh (Figure 15).

At 12 weeks after operation, M1-type macrophages predominated around the surgical mesh in the polypropylene patch group (Table 10, Figure 16 and Figure 17), while there were more M2-type macrophages around the silk fibroin patch . The blood vessel staining of the tissue showed that the number of new blood vessels in the silk fibroin patch group and the mixed patch group was significantly more than that in the polypropylene patch group and the blank control group (p<0.05) (Figure 19), and the new blood vessels mainly gathered at 1 week after operation At the contact surface between the surgical patch and the subcutaneous tissue, the interface between the surgical patch and the abdominal wall muscles, and around the surgical patch, a large number of new blood vessels can be seen growing into the silk fibroin supplement at 4 weeks and 12 weeks after operation. slice (Figure 18).

Table 10 The count of M1 and M2 macrophages in each group at different implantation times (100X) (x±SD)

4. Analysis of Experimental Results

1. Foreign body reaction after patch implantation

Any material implanted in the body will cause the host to respond to the foreign body, called foreign body reaction (FBR), which depends on the characteristics of the material and the susceptibility of the host. FBR is inevitable. An ideal pelvic floor surgical patch should have good biocompatibility. In addition to playing its supporting role, it also needs to be integrated 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.

(1) Protein adsorption

When the material is implanted in the body, the interaction between the wound and the blood material occurs immediately. Within a few seconds, the proteins in the plasma will be adsorbed on the material, forming a very sparse temporary protein matrix of 2~5nm, which is made of fibrin. Mainly, thrombus formation with specific regional differences.

(2) Acute inflammatory response

Acute inflammatory response characterized by polymorphnuclear leukocytes (PMN) (mainly neutrophils, including a small amount of eosinophils and basophils) and mast cell infiltration is the second stage of FBR. It usually lasts from a few hours to a few days, usually resolves within a week, and may become chronic inflammation. Cytokines released by PMN can affect the characteristics and degree of subsequent inflammatory cell recruitment and activation, and thus the phenotype of macrophages during chronic inflammation. According to Brodbeck et al., the initial adhesion of monocytes to the implanted material is short-lived, and the purpose is to send signals to recruit and activate lymphocytes. After lymphocytes release cytokines, monocytes continue to adhere and differentiate into macrophages, and finally differentiate into foreign body giant cells.

(3) Chronic inflammatory response

Chronic inflammatory reactions usually occur within 2–3 weeks of biomaterial implantation, and are characterized by infiltration of lymphocytes and monocytes. Under the influence of various cytokines, monocytes differentiate into macrophages with different phenotypes. The main macrophages exhibit a wound healing phenotype, produce a variety of growth factors to promote cell proliferation and neovascularization, and recruit fibroblasts. The cells form a fibrous capsule that encases the implant, structurally and functionally separating it from the host tissue.

Madden et al. found that the pore size of the implant also significantly affected the chronic inflammation trend of FBR. Cardiac implantation of acellular scaffolds with a pore size of 30-40 µm could find more infiltrating macrophages, and the proportion of M2 macrophages Higher, leading to rapid angiogenic and anti-fibrotic responses, the material integrates best with tissue. Implant stiffness can also affect inflammatory tendencies, as a mismatch between rigid implant materials and lower tissue stiffness around the implant site may lead to higher mechanical stimuli and increased inflammatory responses. If biodegradable materials are implanted, the inflammatory response phase is prolonged, multinucleated giant cells appear, and the collagen production tendency is altered.

(4) Foreign body giant cell formation

The formation of foreign body giant cells (FBG) is considered a hallmark of FBR, which distinguishes FBR from other chronic inflammatory conditions. The formation of FBGC is a process of macrophage fusion, which usually results in the formation of large, multinucleated giant cells tens to hundreds of microns in diameter. And as long as the implant material persists, such as nonabsorbable material, FBGC also exists. Studies have shown that implants with rough surfaces, large surface volumes, grains, and porosity can induce more formation of fibroblasts than implants with smooth, flat surfaces. FBGC.

(5) Fibrous capsule formation

In most cases, the desired outcome is complete integration of the implanted biomaterial into the surrounding tissue after the implant slowly degrades. However, if they cannot be quickly eliminated through phagocytosis or the material is degraded in vivo, macrophages and FBGCs gather around the affected tissue or on the surface of the foreign body, forming a fibrous capsule to isolate and prevent its spread to other body parts. Fibrous capsule formation is influenced by a variety of pro-fibrotic and pro-angiogenic growth factors secreted mainly by M2 macrophages but also by several other cell types such as keratinocytes and fibroblasts.

The effect of implant surface topography, especially porosity, on fibrous capsule formation has been most intensively studied. Numerous studies have concluded that implant surface topography affects early proinflammatory cytokine production and monocyte/macrophage transcription, and that increased implant porosity reduces the thickness of the fibrous capsule formed, reducing fibrosis and Wrapping of the implant material for better integration into the tissue.

The research results of the present invention show that at each treatment time point, more M2 macrophages and more newborns can be observed in the mixed patch group and the silk fibroin patch group than the polypropylene patch group and the blank control group. Angiogenesis. At 1 week after operation, the number of neutrophils in the mixed patch group was the highest, and the inflammatory reaction was the most severe in the acute phase; the silk fibroin patch group and the mixed patch group appeared the earliest and the most foreign body giant cells could be seen. By 12 weeks after operation, the number of neutrophils in the polypropylene patch group was significantly reduced, and only a small amount of lymphocytes were wrapped around the patch, but there were significantly more pro-inflammatory M1 macrophages around the polypropylene patch; silk fibroin Compared with 4 weeks after operation, the lymphocyte counts in the protein patch group and the mixed patch group were significantly reduced, but there were still a large number of foreign body giant cells.

At 12 weeks after the operation, the adhesion scores of the polypropylene mesh group and the mixed mesh group were heavier, and sharp separation was required when taking materials. Tissue staining showed the formation of dense fiber packages around polypropylene. The silk fibroin patch group had less adhesion to the tissue, and tissue staining showed that collagen fibers gradually grew into the patch and wrapped silk fibroin monofilaments.

These differences in inflammatory response and new tissue are related to the material itself and the structural design of the patch. The polypropylene patch has a large monofilament diameter, non-degradable and low-response characteristics, so that inflammatory cells and new collagen fibers are wrapped around the patch, which induces Fibreboard formation, separating tissue from material. However, the degradable properties of silk fibroin in vivo and the small-diameter monofilaments induce macrophages to phagocytize it and the formation of foreign body giant cells, and silk fibroin can be gradually degraded under its action. The small pores between the monofilaments in the silk fibroin patch and the hybrid patch allow the infiltration of inflammatory cells and the rapid growth of collagen fibers, so that the patch can be quickly integrated with the surrounding tissue.

The results of the study showed that the content of type collagen and type III collagen in the pelvic floor connective tissue of POP patients decreased, and the diameter became thinner, which led to poor tissue elasticity and weakened support force, leading to the occurrence of prolapse symptoms.

The material properties, structure and host response of the implanted patch will affect the formation, distribution and typing tendency of collagen fibers. Type III collagen is a newly synthesized collagen with a thin diameter and is more related to tissue elasticity; type I collagen is mainly distributed in mature tissues and has a thicker diameter and is more related to tissue toughness. In the early stage of wound repair, the content of type III collagen increased first, and then gradually replaced by mature type I collagen. This is also similar to the research results of the present invention. The total amount of collagen in the surgical area of each group increases with the prolongation of the implantation time, and the ratio of type I collagen/III collagen is also constantly increasing. At 12 weeks after operation, the silk fibroin patch group and the mixed mesh patch group could induce more type I collagen formation and its enrichment around the patch than the polypropylene patch group. This is also more similar to the fiber composition of the pelvic floor support tissue mainly composed of type I and III collagen. It is speculated that the implantation of silk fibroin patch can better accelerate tissue repair and collagen formation, and the new patch tissue complex can It can better replace and repair the defect or weak tissue of POP patients, support the pelvic floor structure and maintain the function of organs.

The rapid increase in the breaking strength of the silk fibroin patch group and the mixed patch group at 1 week after operation also indicated that silk fibroin can be integrated with the tissue faster after implantation, and the breaking strength of the silk fibroin patch group at 4 weeks after operation The large decrease is due to the degradation of silk fibroin and the destruction of patch structure. The increase of silk fibroin patch at 12 weeks after operation compared with 4 weeks after operation is also related to the growth of new tissue and the integration of the patch, but its long-term in vivo degradation, tissue growth type and distribution trend, and changes in biomechanical properties A longer experimental time is required.

2. Occurrence of patch-related complications

Although fibrosis is necessary during the inflammatory phase, it can also cause mesh-related complications such as patch rupture, erosion, and synechia formation.

(1) Rupture and erosion of patch

Implantation of the surgical mesh triggers a foreign body response, and the persistence of the implant leads to a chronic foreign body response in which macrophages fail to engulf the device and fuse into foreign body giant cells; fibroblasts deposit excess collagen fibers, eventually Wrapping the surgical mesh creates a fragile fibrotic scar. Exacerbated foreign body reaction, persistent inflammation, and subsequent fibrosis are thought to be the main causes of polypropylene patch rupture. To address this issue, the tissue response to the implanted material must be directed toward restoration of normal tissue rather than excessive fibrosis, a process also known as structural remodeling. Structural remodeling can be improved by increasing neovascularization and reducing inflammatory responses.

Mesh-vaginal tissue complexes were excised from 27 women with mesh-related complications, and histological examination revealed a predominance of the M1 subtype of macrophages surrounding the mesh. Persistence of M1 macrophages is thought to lead to chronic inflammation and the formation of foreign body giant cells, and persistent foreign body reactions can lead to tissue fascial erosion injury, mesh displacement and recurrence of organ prolapse. Although the dominant response of M2 macrophages favors tissue integration and ingrowth, excessive M2 macrophage activity also leads to accelerated peri-mesh fibrosis and fibrous capsule formation.

Macrophages are highly plastic and, depending on their environment, can exhibit a wide range of phenotypes and functions. Macrophages of different phenotypes also play very complex roles in the mechanisms of tissue fibrosis and tissue regeneration. If macrophages are depleted in the early stages of body injury, the inflammatory response is usually greatly reduced, which also leads to repair and regeneration. The regeneration efficiency is reduced. The coexistence of pro-inflammatory M1 macrophages and M2 macrophages that promote tissue repair and regeneration can more effectively promote tissue repair and restore homeostasis.

(2) Adhesion

The recommendation and popularity of laparoscopic pelvic floor reconstruction meshes has also brought attention to mesh-related adhesions, caused by fibrinous exudates produced after any form of trauma. These exudates form a temporary adhesion until the fibrinolytic system absorbs the fibrin. In the presence of ischemia, inflammation, or foreign bodies (such as patches), fibrin absorption can be delayed and tissue adhesions can even form.

When the patch is placed in the vicinity of the bowel, all the patches will adhere, and the extent of the adhesion depends on the size of the pore size, filament structure and surface area of the patch. Heavier patches induce a strong fibrotic response, ensuring firm adhesion to surrounding tissue, as well as dense adhesion. In contrast, microporous patches do not allow tissue ingrowth and have a very low risk of adhesions, but the patch itself does not adhere firmly to the abdominal wall.

(3) infection

The risk of infection mainly depends on the type of filament used and the pore size, microporous patches have a higher risk of infection due to the inability of macrophages and neutrophils to enter the small pores (<10 µm), which only allow bacteria ( <1µm) survive within the mesh. Similar problems can also occur with multifilament patches. Therefore, the meshes with the lowest risk of infection are those made of monofilaments with pores larger than 75 µm, which eliminate the need for removal of the infection.

The main pore diameters of the three patches used in this study are all > 200 μm, which can meet the needs of immune inflammatory response and new tissue growth, but the existence of small pores between silk fibroin monofilaments may increase the risk of infection. The stiffness of polypropylene mesh and hybrid mesh is significantly greater than silk fibroin mesh and pelvic floor mechanics, and there are risks of disuse atrophy of surrounding tissues and exposure of mesh. As of 12 weeks after the operation, no complications such as infection, patch exposure, and erosion have been observed in the experimental rats.

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

1. Due to the existence of polypropylene monofilaments, the hybrid patch still maintains similar mechanical properties to the polypropylene patch group after 12 weeks after surgery; the silk fibroin patch degrades after implantation, Integrated into the surrounding tissue, the fracture strength and stiffness of the formed patch-tissue complex are more similar to those of the adjacent tissue, which can meet the needs of pelvic floor mechanics.

2. There are more M2 macrophages, foreign body giant cells and new blood vessels around the silk fibroin patch and mixed patch; M1 macrophages dominate around the polypropylene patch, and a small amount of foreign body giant cells can be seen. cells, indicating the possibility of long-term chronic inflammation.

3. The silk fibroin patch group and the mixed patch group can induce more new collagen, and the proportion of type I collagen/type III collagen in the patch tissue complex is closer to that of the normal basin at 12 weeks after operation. Bottom tissue. Collagen fibers quickly grow into the patch and wrap silk fibroin monofilaments, and the integration of the patch with the tissue is better than that of the polypropylene patch.

Claims (3)

1. A preparation method for a silk fibroin patch for the treatment of women's pelvic floor dysfunction, characterized in that: comprising the steps: The silk fibroin thread is warp-knitted and then heat-set; The diameter of the silk fibroin thread is 120-125 µm; The conditions of the warp knitting are as follows: Laying yards are GB1:21/23/10/12/10/23:21//23//, GB2:00/00/33/33/00/00/33/33//, GB3:12/ 10/23:21/23/10/12/10//; The threading method is one thread and one empty; The let-off amounts are 2250 mm/rack, 850 mm/rack and 2250 mm/rack respectively; Using RS4EL warp knitting machine for warp knitting; The heat-setting conditions are as follows: the temperature is 130° C., and the time is 6-8 minutes. 2. the silk fibroin patch prepared by the method described in claim 1; The pore diameter of the silk fibroin patch is >200µm, the surface density is 51.16±0.16g/m ² , and the porosity is 39.09±0.25%. 3. The application of the silk fibroin patch according to claim 2 in the preparation of the pelvic organ prolapse patch.

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