Preparation method and application of absorbable bioactive suture line with tissue repair function

Abstract

The invention discloses a preparation method of an absorbable bioactive suture line with a tissue repair function, which comprises the following steps: activating the surface of a PGA-PCL suture line by using polynoradrenaline, and then assembling human umbilical cord mesenchymal stem cell exosome to the surface of the activated suture line by using a soaking method to construct the absorbable suture line with surface functionalization. After the suture line is used, the wound healing effect is good, the blood vessel and cell recovery degree is good, proliferation and migration of fibroblasts can be remarkably promoted, proliferation of T lymphocytes and Th1 cells is effectively inhibited, and a powerful guarantee is provided for tissue repair.

Description

Technical Field

[0001] The invention relates to a preparation method and application of an absorbable bioactive suture with tissue repair function, belonging to the technical field of medical biomaterials. Background Art

[0002] In modern medicine, sutures are an indispensable tool in surgery, and their performance is directly related to the quality and speed of wound healing. PGA-PCL sutures are bioresorbable materials made by copolymerization of polyglycolic acid (PGA) and polycaprolactone (PCL). This material combines the advantages of both polymers and has the advantages of high strength, moderate flexibility, and controllable degradation rate. This suture gradually degrades in the body and eventually turns into carbon dioxide and water, avoiding the need for secondary surgery for patients. PGA-PCL sutures are widely used in tissue repair due to their good biocompatibility and bioresorbability.

[0003] However, with the development of medical technology and the increase in patient needs, PGA-PCL suture materials still have some limitations, including varying degrees of swelling at the implant site, which can cause an acute inflammatory response in the early stages of degradation. Although the swelling can be eliminated by draining the fluid from the swollen area, it also affects wound healing and the patient's experience; the degradation rate of PGA-PCL sutures can theoretically be controlled by adjusting the ratio of PGA and PCL, but in actual applications the control of the degradation rate may not be as expected and may not meet specific wound healing requirements; in addition, the lack of cell recognition sites on the surface of PGA and PCL may hinder cell adhesion to the sutures, and the lack of the ability to promote tissue regeneration limits its application in the treatment of complex wounds.

[0004] Surface modification of materials is a very effective technical means. In recent years, research on introducing reactive functional groups on the surface of materials based on catecholamine surface chemical modification methods has become a hot topic. Studies have found that the presence of side chain amine groups in the structure of catecholamines makes their self-polymerization ability relatively strong, so it can form a stable coating by polymerizing itself as a monomer, among which dopamine (DA) is the most widely used. DA uses the strong adhesion and coordination ability of the o-phenol group and the Michael addition and Schiff base reaction of the lysine and quinone groups with double-terminal amine groups to give the system the ability to self-polymerize in the form of covalent bonds.

[0005] Noradrenaline (NE) is a catecholamine biomolecule with a chemical structure similar to DA. It has a structure of catechol and terminal amine groups. It can be used as a monomer to form a stable coating through various effects such as coordination bonds, π-π conjugation, oxidative self-polymerization, and electrostatic assembly. In addition, there is an additional hydroxyl group on the ortho-carbon of the side chain amine group, which gives it a different ability from DA. At the same time, the presence of the side chain hydroxyl group also gives the poly-noradrenaline (pNE) coating a stronger ring-opening polymerization ability, which can initiate more reactions. In addition, the pNE coating does not have the quantitative interference of ligands and the aggregation of micro-nanoparticles in the pDA coating, resulting in the high surface roughness of the coating. It is very suitable for loading drug molecules, peptides, proteins, and various factors, and is considered to be a multifunctional surface functionalization platform. Therefore, constructing a catecholamine coating is an effective modification strategy.

[0006] Exosomes are membrane vesicles with a diameter of 50 to 200 nm, containing RNA, lipids and proteins, and can participate in intercellular communication. Umbilical cord mesenchymal stem cell exosomes (Hu-MSC-Exo) have the advantages of tissue repair and immunomodulatory functions, as well as non-immunogenicity and non-tumorigenicity. As an emerging therapeutic method, mesenchymal stem cell exosomes significantly improve wound healing and accelerate the repair of damaged tissues by regulating inflammatory responses, promoting the proliferation and migration of fibroblasts, and promoting angiogenesis. As an extracellular vesicle, mesenchymal stem cell exosomes can not only be loaded on the surface of the coating to achieve a sustained release function and improve the stability and utilization of exosomes, but also have the advantages of long directional duration.

[0007] In view of the problems of current absorbable sutures, such as single drug carrier function, poor suture operation feel and limited healing function, there is an urgent need to improve the existing absorbable sutures to better meet the clinical needs of sutures in wound healing. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides an absorbable suture containing mesenchymal stem cell exosomes and a preparation method thereof.

[0009] The technical solution adopted by the present invention is:

[0010] A method for preparing an absorbable bioactive suture with tissue repair function, comprising the following steps:

[0011] (1) Preparation of umbilical cord mesenchymal stem cell exosomes Hu-MSC-Exo: First-generation human umbilical cord mesenchymal stem cells were inoculated into cell culture flasks, with 1.2×10 6The cells were placed in a carbon dioxide incubator for culture and observed under a microscope. When the cell confluence reached 80%, the culture medium was aspirated, the cells were washed with PBS, and serum-free culture medium was added. The cells were placed in a carbon dioxide incubator for another 24 hours, and the supernatant was collected. Hu-MSC-Exo was collected by ultracentrifugation.

[0012] The ultracentrifugation method is as follows: the collected supernatant is placed in a centrifuge tube, and centrifuged at 300×g for 10 minutes, 2000×g for 10 minutes, and 10000×g for 30 minutes at 4°C to remove dead cells and debris; then ultracentrifuged at 100000×g for 70 minutes at 4°C, the supernatant is discarded, and the supernatant is resuspended with a small amount of PBS, and ultracentrifuged again at 100000×g for 70 minutes, and the supernatant is discarded to obtain Hu-MSC-Exo.

[0013] (2) Preparation of norepinephrine-polyglycolic acid-polycaprolactone suture: dissolve norepinephrine NE in Tris buffer, then add polyglycolic acid-polycaprolactone PGA-PCL suture, let it soak in a sterile and dust-free environment at 25°C for 12 hours, take out the suture, ultrasonically clean it with sterile distilled water, dry it with nitrogen and sterilize it to obtain norepinephrine-modified pNE-PGA-PCL suture;

[0014] (3) Preparation of exosome-loaded norepinephrine-polyglycolic acid-polycaprolactone sutures: Sterile pNE-PGA-PCL sutures were placed in an exosome solution and allowed to stand in a 37°C constant temperature incubator for 1 hour to adsorb exosomes; the exosome-absorbed sutures were then placed in a vacuum freeze dryer and vacuum freeze-dried at -75°C for 48 hours to obtain pNE-PGA-PCL-Exo sutures, with an exosome solution concentration of 2.5 to 4.5 mg / mL.

[0015] The absorbable bioactive suture prepared by the method can be used in wound suture.

[0016] Beneficial effects of the present invention:

[0017] (1) The present invention utilizes norepinephrine to modify the existing PGA-PCL suture, and through functional modification of the suture surface, umbilical cord mesenchymal stem cell exosomes are loaded onto the PGA-PCL suture to construct a new type of exosome-loaded absorbable bioactive suture.

[0018] (2) The present invention loads human umbilical cord mesenchymal stem cell exosomes onto the surface of activated sutures by immersion method, which can effectively reduce inflammatory response, promote cell proliferation, and improve cell migration ability and tissue regeneration ability.

[0019] (3) In vitro cell experiments have shown that the exosome-loaded pNE functional coating can effectively promote the proliferation and migration of fibroblasts, increase the inhibition rate of T lymphocytes and Th1 cells, inhibit local inflammatory responses, promote collagen deposition and angiogenesis, improve tissue regeneration ability, provide protection for tissue repair, and provide new ideas for the development of bioactive sutures.

[0020] (4) The preparation method of the present invention is simple, practical and easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Transmission electron microscopy image of umbilical cord mesenchymal stem cell exosomes;

[0022] Figure 2 Particle size analysis of umbilical cord mesenchymal stem cell exosomes;

[0023] Figure 3 Western blot of umbilical cord mesenchymal stem cell exosomes;

[0024] Figure 4 Cryo-EM images of PGA-PCL-Exo sutures and pNE-PGA-PCL-Exo sutures;

[0025] Figure 5 Exosome release rates of different sutures;

[0026] Figure 6 The proliferation rate of fibroblasts after culturing different sutures with fibroblasts for 1 and 3 days;

[0027] Figure 7 The inhibition rate of T cells after 5 days of culture of different sutures with T cells;

[0028] Figure 8 The proportion of Th1 cell population after culturing different sutures with Th1 cells for 6 hours;

[0029] Fig. 9 Wound healing of different suture lines on days 0, 3, and 7 after surgery;

[0030] Fig.10 H&E staining results of wound tissues 3 and 7 days after surgery using different sutures;

[0031] Fig.11 Mssson staining results of wound tissues 7 days after surgery with different sutures;

[0032] Fig.12 CD31 immunohistochemistry images of wound tissues 3 and 7 days after surgery with different sutures;

[0033] Fig.13Ki67 immunohistochemistry images of wound tissues 3 and 7 days after surgery with different sutures;

[0034] Fig.14 CD68 immunohistochemistry of wound tissues 3 and 7 days after surgery with different sutures. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments.

[0036] Cells, materials and main reagents that may be used in the present invention:

[0037] Human umbilical cord mesenchymal stem cells Hu-MSCs, human monocytic leukemia cells THP-1, human peripheral blood mononuclear cells PBMCs, human T lymphocytes, human Th1 cells, human umbilical cord mesenchymal stem cell exosomes Hu-MSC-Exo.

[0038] Culture of Hu-MSCs: using human umbilical cord mesenchymal stem cell serum-free culture medium (Youkang Biotechnology (Beijing) Co., Ltd.); culture of human fibroblasts: using DMEM / F12 (containing 10% fetal bovine serum and 1% penicillin-streptomycin) complete culture medium; culture of THP-1, human Th1 cells and T lymphocytes: using RPMI-1640 (containing 10% fetal bovine serum and 1% penicillin-streptomycin) complete culture medium; DMEM / F12, RPMI-1640, fetal bovine serum and penicillin-streptomycin were purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0039] CD9 antibody, TSG101 antibody, and Calnexin antibody were purchased from Cell Signaling Technology, β-actin internal reference antibody was purchased from Beijing Bioson Biotechnology Co., Ltd., CD3 antibody, CD8 antibody, and IFN-γ antibody were purchased from BD, and Micro BCA TM Protein detection kit (23235), cell stimulation mixture (with protein transport inhibitor) (00-4957-03), and intracellular fixation permeabilization buffer (88-8824-00) were purchased from Thermo Fisher Scientific, CCK-8 was purchased from Dongren Chemical Technology (Shanghai) Co., Ltd., and norepinephrine and kidney bean lectin PHA-M (L2642) were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0040] Example 1 Preparation of a mesenchymal stem cell exosome-loaded absorbable suture

[0041] The preparation method comprises the following steps:

[0042] 1. Preparation and identification of human umbilical cord mesenchymal stem cell exosomes (Hu-MSC-Exo)

[0043] 1.1 Preparation of Hu-MSC-Exo: First generation Hu-MSCs were inoculated in T175cm 2 Cell culture flask, 1.2×10 6 The cells were placed in a carbon dioxide incubator for culture. When the cell confluence reached 80% under a microscope, the culture medium was aspirated, and the cells were washed three times with 20 mL of sterile PBS and supplemented with serum-free culture medium. The cells were placed in a carbon dioxide incubator for another 24 hours, and the supernatant was collected. Hu-MSC-Exo was collected by ultracentrifugation.

[0044] Collection of Hu-MSC-Exo: The collected supernatant was placed in a centrifuge tube and centrifuged at 300×g for 10 minutes, 2000×g for 10 minutes, and 10000×g for 30 minutes at 4°C to remove dead cells and debris. Then, an ultracentrifuge was used to centrifuge at 100000×g for 70 minutes at 4°C; the supernatant was discarded, resuspended with a small amount of sterile PBS, and centrifuged again at 100000×g for 70 minutes, and the supernatant was discarded to obtain human umbilical cord mesenchymal stem cell exosomes, which were suspended with 500μL sterile PBS and stored at -80°C for later use.

[0045] 1.2 Identification of Hu-MSC-Exo: The International Extracellular Vesicles Association recommends that the identification of Hu-MSC-Exo should detect at least three types of proteins: transmembrane protein CD9, cytosolic protein TSG101, and negative protein Calnexin; and observe the morphology, concentration, and size distribution of exosomes to demonstrate the characteristics of exosomes.

[0046] Western blot was used to detect the expression of CD9, TSG101, and Calnexin in exosomes: RIPA lysis buffer was added to Hu-MSC-Exo, and the concentration was determined using a protein quantitative detection kit. SDS-PAGE gel electrophoresis was then performed, and after transfer and blocking, CD9, TSG101, and Calnexin primary antibodies were added respectively and incubated at 4°C overnight. After washing the membrane with TBST, β-actin antibody was added and incubated for 1 hour, the membrane was washed, and the image was exposed.

[0047] The morphological characteristics and population characteristics of exosomes were analyzed by transmission electron microscopy and nanoparticle tracking technology (completed by Nanjing Zhongjing Science and Technology Co., Ltd.). Transmission electron microscopy observation steps: 10 μL Hu-MSC-Exo was dropped on the carbon support film sample copper mesh, left at room temperature for 2 minutes, and the liquid was absorbed from the side of the filter with filter paper, and negatively stained with 3% phosphotungstic acid solution at room temperature for 5 minutes, and the negative staining liquid was absorbed by filter paper, dried at room temperature, and observed and photographed with electron microscopy.

[0048] Nanoparticle tracking analysis steps: Take Hu-MSC-Exo out of the -80℃ freezer, place it in an ice box, thaw and centrifuge. Take 20uL and add it to a 1.5mL EP tube, then add 980uLDPBS buffer to dilute it 50 times, use a 1mL syringe to load the diluted solution, observe the particle image on the screen, adjust the focus and parameters, and start the detection.

[0049] The results are as follows Figure 1 to Figure 3 As shown, Figure 1 Transmission electron microscopy results showed that Hu-MSC-Exo had a cup-shaped structure with a size of 50 to 200 nm; Figure 2 Nanoparticle tracking analysis showed that the size of exosomes was mainly distributed between 50 and 200 nm; Figure 3 Western blotting results confirmed that Hu-MSC-Exo expressed the transmembrane protein CD9 and the cytosolic protein TSG101, and did not express the negative protein Calnexin; through the characterization of morphological dimensions, particle size distribution dimensions and protein dimensions, it was confirmed that the obtained component was Hu-MSC-Exo.

[0050] 2. Preparation of Exosome-loaded pNE-PGA-PCL Sutures

[0051] Due to the presence of side chain amine groups, norepinephrine can self-polymerize onto the material surface in Tris buffer, so the polynorepinephrine (pNE) coating was prepared by a "one-step dip coating method".

[0052] NE was dissolved in Tris buffer at pH = 8.5. After it was completely dissolved, PGA-PCL suture was added and allowed to soak in a sterile, dust-free environment at 25°C for 12 hours. The suture was then taken out and ultrasonically cleaned 3 times with sterile distilled water and dried with nitrogen to obtain NE-modified PGA-PCL suture, i.e., pNE-PGA-PCL suture, which was sterilized with ethylene oxide for later use.

[0053] The sterile pNE-PGA-PCL suture was placed in an exosome solution with a concentration of 2.5-4.5 mg / mL and placed in a 37°C incubator for 1 hour to adsorb the exosomes.

[0054] 3. Suture Drying

[0055] The exosome-absorbed suture was placed in a vacuum freeze dryer and vacuum freeze-dried at -75°C for 48 hours to obtain the exosome-loaded pNE-PGA-PCL suture, i.e., pNE-PGA-PCL-Exo suture, which was stored in a -80°C refrigerator for future use.

[0056] Example 2 Performance and application of mesenchymal stem cell exosome-loaded absorbable sutures

[0057] The pNE-PGA-PCL-Exo suture of Example 1 was subjected to performance testing to further study its related applications.

[0058] 1. Mechanical properties test of different sutures

[0059] Referring to the YY0167-2020 and YY1116-2020 standards, the tensile properties of the pNE-PGA-PCL-Exo sutures were tested. The test was divided into a pNE-PGA-PCL-Exo suture group and a PGA-PCL suture group. The PGA-PCL suture was used as a control, with 3 sutures in each group, and the test was performed 3 times. The sutures were fixed on a multifunctional strength meter, the tensile speed of the specimen was set to 300 mm / min, the test gauge length was 130 mm, and the strength meter applied tension until the suture broke, and the maximum tension value was recorded as the breaking strength value. The results are shown in Table 1.

[0060] The results showed that compared with PGA-PCL sutures, the breaking strength and breaking elongation of pNE-PGA-PCL-Exo sutures were improved. It may be that the interaction between the norepinephrine coating and the suture material changed the original surface morphology, thus affecting the mechanical properties of the suture.

[0061] Table 1 Mechanical properties of different sutures

[0062]

[0063] 2. Surface morphology of different sutures

[0064] To observe whether exosomes were adsorbed to the surface of pNE-PGA-PCL-Exo sutures, a cold field emission scanning electron microscope (Hitachi, Regulus8100) was used to observe the surface state of the sutures, and PGA-PCL-Exo sutures were used as a control.

[0065] The results are as follows Figure 4 As shown, compared with PGA-PCL sutures, pNE-PGA-PCL sutures have more particles adsorbed on their surface, which are exosomes, indicating that the norepinephrine coating changes the original surface morphology of the sutures and adsorbs more exosomes.

[0066] 3. Exosome release rate of different sutures

[0067] The PGA-PCL-Exo sutures and pNE-PGA-PCL-Exo sutures were immersed in sterile PBS and placed in a 37°C incubator for 0.25 days, 1 day, 3 days, 5 days, 7 days, 10 days, 13 days, and 15 days, respectively, and the protein concentration in the solution at each time point was detected.

[0068] The results are as follows Figure 5 As shown, both groups of sutures released exosomes rapidly in the first 3 days and were able to release them stably and continuously. The exosome release capacity of the pNE-PGA-PCL-Exo suture was significantly higher than that of the PGA-PCL-Exo suture, indicating that the pNE coating adsorbed more exosomes, indicating that the scheme of constructing an exosome release surface with the pNE coating is a controllable release strategy.

[0069] 4. Evaluation of the proliferation ability of different sutures on fibroblasts

[0070] Whether it is the surface skin or the internal tissue, it is inevitable to come into contact with fibroblasts, so the compatibility evaluation of sutures and fibroblasts is particularly important.

[0071] PGA-PCL sutures, PGA-PCL-Exo sutures, pNE-PGA-PCL sutures, and pNE-PGA-PCL-Exo sutures were placed in 6-well plates, and fibroblasts were cultured at a density of 1×10 5 The cells / well were added to the wells of different sutures and cultured in a carbon dioxide incubator for 3 days.

[0072] The CCK-8 reagent with a concentration of 10% was prepared in DMEM / F12 culture medium under light-proof conditions, and the proliferation rate of fibroblasts after culturing for 1 day and 3 days was detected using the CCK-8 reagent.

[0073] CCK-8 detection: aspirate the culture medium in the well plate, wash once with sterile PBS, then add 350 μL of 10% CCK-8 reagent to each well, place in a carbon dioxide incubator for 3 hours, aspirate 200 μL of reaction solution from each well and add it to a 96-well plate, use an enzyme reader to detect the absorbance value at a wavelength of 450 nm, and calculate the cell proliferation rate. Cell proliferation rate = (absorption value of the experimental group - absorption value of the blank control) / (absorption value of the control group - absorption value of the blank control) × 100%.

[0074] The results are as follows Figure 6 As shown in the figure, after the introduction of pNE coating and Hu-MSC-Exo, the proliferation rate of cells in the PGA-PCL suture group was the lowest, and the other three groups significantly promoted the growth of fibroblasts, and the proliferation rate of the group that introduced pNE and Hu-MSC-Exo at the same time was the highest, indicating that the constructed pNE-PGA-PCL-Exo suture has a more significant effect on promoting fibroblasts. The asterisks in the figure indicate that *p<0.05, **p<0.01, ***p<0.001.

[0075] 5. Evaluation of the inhibitory ability of different sutures on T lymphocytes

[0076] PBMCs were cultured at 1 × 10 6Cells / well were added to a 6-well plate, and 10 μg / mL of PHA-M was added to each well to stimulate lymphocyte proliferation. PGA-PCL sutures, PGA-PCL-Exo sutures, pNE-PGA-PCL sutures, and pNE-PGA-PCL-Exo sutures were placed in the wells, respectively, and placed in a carbon dioxide incubator for 5 days. The absorbance value at a wavelength of 450 nm was detected by CCK-8 using an ELISA reader to calculate the T lymphocyte proliferation inhibition rate. T lymphocyte proliferation inhibition rate = (1-positive cells in the experimental group / positive control group cells) × 100%.

[0077] The results are as follows Figure 7 As shown: the pNE-PGA-PCL-Exo suture group has the strongest inhibitory ability on T lymphocytes, with an inhibition rate of 88.45%, and the PGA-PCL suture group has the worst inhibitory ability on T lymphocytes, with an inhibition rate of 71.99%. This shows that the sutures modified with pNE coating and adsorbed with Hu-MSC-Exo can significantly inhibit the proliferation of T lymphocytes and reduce local inflammatory responses.

[0078] 6. Evaluation of the inhibitory ability of different sutures on Th1 cells

[0079] Th1 cells play an important role in the wound healing process, so it is necessary to evaluate the inhibitory ability of pNE-PGA-PCL-Exo sutures on Th1 cells.

[0080] PBMCs were cultured at 1 × 10 6 Cells / well were added to a 6-well plate and cultured in a carbon dioxide incubator for 1 day. Then, PGA-PCL sutures, PGA-PCL-Exo sutures, pNE-PGA-PCL sutures, and pNE-PGA-PCL-Exo sutures were placed in the well plate, and a cell stimulation mixture was added and cultured for another 6 hours.

[0081] Collect cells in a flow tube, centrifuge at 500×g for 8 minutes, discard the supernatant, and vortex to completely dissociate the cell clusters; add 5μL of CD3 and CD8 antibodies respectively, vortex to mix, incubate at room temperature in the dark for 30 minutes, centrifuge at 500×g for 8 minutes, and discard the supernatant. Add 100μL of fixative, vortex to mix, and incubate at room temperature in the dark for 30 minutes. Add 2mL of permeabilization solution, centrifuge at 500×g for 8 minutes, and discard the supernatant (repeat once). Resuspend the cell pellet in 100μL of permeabilization solution, add 5μL of IFN-γ antibody, vortex to mix, and incubate at room temperature in the dark for 30 minutes. Add 2mL of permeabilization solution, centrifuge at 500×g for 8 minutes, and discard the supernatant (repeat once). Add 500μL of PBS to resuspend the cells, and use a flow cytometer (BD, Canto plus) to detect CD3 + CD8 - IFN-γ+ proportion of cell populations.

[0082] The results are as follows Figure 8 As shown: the proportion of Th1 cells in the PGA-PCL suture group, PGA-PCL-Exo suture group, pNE-PGA-PCL suture group, and pNE-PGA-PCL-Exo suture group were 10.5%, 3.59%, 3.93%, and 1.83%, respectively. Compared with the PGA-PCL suture group, the sutures modified with pNE coating and Hu-MSC-Exo can significantly inhibit the proliferation of Th1 cells, and the pNE-PGA-PCL-Exo suture group has the strongest inhibitory ability on Th1 cells. The results show that the sutures after the pNE coating adsorbed Exo can significantly inhibit the local inflammatory response.

[0083] Example 3 Application of pNE-PGA-PCL-Exo suture

[0084] 1. Wound repair experiment

[0085] A full-thickness skin defect model was used to investigate the effects of sutures on wound healing and regeneration. All sutures were sterilized with ethylene oxide and the surgical instruments involved were sterilized with high temperature and high pressure.

[0086] Female SD rats aged 6-8 weeks were anesthetized and their back hair was shaved. Then, full-thickness skin defect models with a length of 2 cm were created on both sides of the rats' backs. The wounds were sutured with PGA-PCL sutures and pNE-PGA-PCL-Exo sutures, respectively, and a repair comparison experiment was performed.

[0087] After suturing, the rats were returned to the cages. During the experiment, the rats were observed every day for infection, death, and other problems, and the results were recorded. The rats were in good health during the experiment, and no obvious inflammation or infection was observed in the wounds repaired by sutures in the two groups.

[0088] Photos of the back wounds of rats at 0, 3, and 7 days after trauma Fig. 9 Judging from the wound photos, the wound healing of the pNE-PGA-PCL-Exo suture group was better, especially on the 7th day, there were fewer scars and some new hair.

[0089] 2. H&E staining of postoperative wound tissue

[0090] H&E staining was used to analyze the inflammatory response near the wound on the 3rd and 7th days after surgery. Fig.10As shown, whether it is 3 days or 7 days after surgery, the epidermis of the wound skin tissue in the pNE-PGA-PCL-Exo suture group is relatively intact, the structure of each layer is clear, the dermis is thick, the number of skin appendages, hair follicles and sebaceous glands are abundant, the structure is clear, and the distribution is scattered. Fibroblast proliferation and neovascularization can be seen locally; compared with the pNE-PGA-PCL-Exo suture group, the number of samples with fibroblast proliferation and inflammatory cell infiltration in the dermis and subcutaneous tissue of the PGA-PCL suture group increased, the proliferation area was wide, the inflammatory cell infiltration was severe, the number of samples of cerebral stratum cavitation increased, and the degree was severe, and the number of samples of myocyte necrosis and multinucleated giant cell infiltration increased. This result clearly shows that pNE-PGA-PCL-Exo has a significant promoting effect on early wound healing and can significantly improve the inflammatory response during wound healing.

[0091] 3. Collagen regeneration in postoperative wounds

[0092] Collagen is the main structural protein of the extracellular matrix of the dermis. The synthesis and deposition of collagen are essential for restoring the function and strength of tissues. Masson staining was performed on the wound tissue on the 7th day to compare the effects of collagen regeneration in the skin defect site of the suture line between the two groups, and the staining pictures were obtained (collagen was stained blue, keratin and muscle fibers were stained red).

[0093] Masson staining results Fig.11 As shown, ImageJ software was used to calculate the collagen volume fraction (the percentage of the collagen-positive blue area to the total tissue area). On the 7th day, the PGA-PCL suture group had only a small amount of collagen regeneration, with a positive area ratio of 29.4557±10.5018%, while the pNE-PGA-PCL-Exo suture group had a large amount of collagen generation, with a positive area ratio of 38.7838±4.6783%, which was 1.32 times that of the PGA-PCL suture group (see Table 2). This result shows that exosomes have a significant promoting effect on early collagen regeneration and wound healing, which is consistent with the results of H&E, and the pNE-PGA-PCL-Exo suture group has more complete skin appendages, indicating that the pNE-PGA-PCL-Exo suture has a better ability to promote tissue repair.

[0094] Table 2 Positive area ratio of two groups of sutures

[0095]

[0096] 4. Postoperative Angiogenesis

[0097] Angiogenesis plays a multifaceted role in wound healing, from hemostasis to tissue remodeling. Angiogenesis is indispensable. CD31, as a marker of vascular endothelial cells, plays an important role in angiogenesis and repair processes. In immunohistochemistry, the staining intensity of CD31 can be used to evaluate the formation of new blood vessels.

[0098] To verify the angiogenesis-promoting ability of pNE-PGA-PCL-Exo sutures, CD31 immunohistochemistry was used to analyze the angiogenesis of the wound site on days 3 and 7, and ImageJ software was used to calculate the percentage of immunohistochemical positive signals. Fig.12 As shown, for the PGA-PCL suture group, although the positive expression of CD31 increased from the 3rd day to the 7th day, there were only a few tiny blood vessels, which were weakly positively expressed (+). Compared with the PGA-PCL suture group, the pNE-PGA-PCL-Exo suture group had a stronger ability to promote angiogenesis, especially on the 7th day, the epidermis and sebaceous glands and hair cysts in the dermis, the fibrous connective tissue in the dermis and subcutaneous tissue, and the smooth muscle of the muscular layer in the pNE-PGA-PCL-Exo suture group were strongly positively expressed (+++), indicating that the pNE-PGA-PCL-Exo suture has a stronger synergistic effect with angiogenesis and can significantly promote angiogenesis during wound healing.

[0099] Positive expression is divided into three levels: weakly positive (+), moderately positive (++) and strongly positive (+++). Moderately positive refers to a positive cell ratio of 25% to 49%, and strongly positive refers to a positive cell ratio of more than 50%.

[0100] 5. Postoperative wound cell proliferation

[0101] Cell proliferation is a key link in the wound epithelialization and healing process, and it is closely related to wound repair and regeneration. If cell proliferation is inhibited or uncoordinated, it will lead to delayed wound healing or the formation of undesirable scars. Immunohistochemical detection of Ki67 expression can be used to assess the activity of cell proliferation. The higher the Ki67 index, the more cells are in a proliferative state.

[0102] The Ki67 immunohistochemistry method was used to detect the expression of Ki67 in wound tissues on days 3 and 7. The percentage of immunohistochemical positive signals was calculated using ImageJ software. The results are shown in the figure. Fig.13As shown. With the extension of wound healing time, the positive expression of Ki67 gradually increased. On the 3rd day, both the pNE-PGA-PCL-Exo suture group and the PGA-PCL suture group were weakly positively expressed (+); on the 7th day, the epidermis, sebaceous glands and hair cysts in the dermis, and smooth muscle of the muscular layer of the pNE-PGA-PCL-Exo suture group were strongly positively expressed (+++), while the PGA-PCL suture group was still weakly positively expressed (+). This shows that PGA-PCL sutures can also promote cell proliferation, but it is relatively slow, while pNE-PGA-PCL-Exo sutures can quickly promote cell proliferation in the early stage of wound healing.

[0103] 6. Expression of macrophages in postoperative wounds

[0104] During wound healing, high expression of CD68 is usually associated with increased inflammatory activity. In immunohistochemistry, CD68 expression is usually used to identify and quantify macrophages in tissues. ImageJ software was used to calculate the percentage of immunohistochemical positive signals. CD68 immunohistochemical analysis of macrophage expression in wound sites on days 3 and 7 was performed. The results are shown in Figure 2. Fig.14 As shown. No positive expression area was found in the pNE-PGA-PCL-Exo suture group on either the 3rd or 7th day, while moderate positive expression (++) was found in the cytoplasm of the dermis and subcutaneous tissue of the PGA-PCL suture group. The results showed that the pNE-PGA-PCL-Exo suture can control the inflammation that occurs during wound healing, reduce the number of macrophages, and significantly reduce the inflammatory response of tissues.

Claims

1. A method for preparing an absorbable bioactive suture with tissue repair function, characterized in that: The following steps are involved: (1) Preparation of umbilical cord mesenchymal stem cell exosomes Hu-MSC-Exo: First-generation human umbilical cord mesenchymal stem cells were inoculated into cell culture flasks, with 1.2×10 6 The cells were placed in a carbon dioxide incubator for culture and observed under a microscope. When the cell confluence reached 80%, the culture medium was aspirated, the cells were washed with PBS, and serum-free culture medium was added. The cells were placed in a carbon dioxide incubator for another 24 hours, and the supernatant was collected. Hu-MSC-Exo was collected by ultracentrifugation. (2) Preparation of norepinephrine-polyglycolic acid-polycaprolactone suture: dissolve norepinephrine NE in Tris buffer, then add polyglycolic acid-polycaprolactone PGA-PCL suture, let it soak in a sterile and dust-free environment at 25°C for 12 hours, take out the suture, ultrasonically clean it with sterile distilled water, dry it with nitrogen and sterilize it to obtain norepinephrine-modified pNE-PGA-PCL suture; (3) Preparation of exosome-loaded norepinephrine-polyglycolic acid-polycaprolactone sutures: Sterile pNE-PGA-PCL sutures were placed in an exosome solution and allowed to stand in a 37°C constant temperature incubator for 1 hour to adsorb exosomes; the exosome-absorbed sutures were then placed in a vacuum freeze dryer and vacuum freeze-dried at -75°C for 48 hours to obtain pNE-PGA-PCL-Exo sutures.

2. The method for preparing the absorbable bioactive suture according to claim 1, characterized in that: The ultracentrifugation method is as follows: the collected supernatant is placed in a centrifuge tube, and centrifuged at 300×g for 10 minutes, 2000×g for 10 minutes, and 10000×g for 30 minutes at 4°C to remove dead cells and debris; then ultracentrifuged at 100000×g for 70 minutes at 4°C, discarded the supernatant, resuspended with a small amount of PBS, and ultracentrifuged again at 100000×g for 70 minutes, discarded the supernatant, and obtained Hu-MSC-Exo.

3. The method for preparing the absorbable bioactive suture according to claim 1, characterized in that: The concentration of the exosome solution is 2.5 to 4.5 mg / mL.

4. Use of the absorbable bioactive suture prepared by the method according to claim 1 in wound suturing.

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