Preparation method and application of absorbable suture line loaded with mesenchymal stem cell exosome

Abstract

The invention discloses a preparation method and application of a mesenchymal stem cell exosome-loaded absorbable suture line, noradrenaline is utilized to activate the surface of a collagen suture line, then human umbilical cord mesenchymal stem cell exosomes are assembled to the surface of the activated suture line through a soaking method, and the absorbable suture line with the surface functionalization is constructed. 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 suture thread loaded with mesenchymal stem cell exosomes, 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. The ingredients of collagen sutures are mainly extracted from animal collagen. Through processes such as removing impurities and cross-linking, a stable structure is formed between collagen molecules, thereby enhancing the strength and tensile properties of the sutures. Over time, they are gradually degraded and absorbed in the body, reducing the risk of secondary damage and infection caused by suture removal.

[0003] Although collagen sutures have some advantages in physicochemical and biological aspects, they still have some limitations, including individual allergic reactions caused by immunogenicity, inflammatory reactions caused by cross-linking agents stimulating surrounding tissues during processing, changes in local tissue osmotic pressure in special tissues (such as nerve tissue) leading to abnormal symptoms such as local numbness, and bleeding and cracking of wounds due to rapid degradation and absorption in vascular-rich tissues, which limits the application of collagen sutures.

[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 its 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] As an emerging treatment method, mesenchymal stem cell exosomes can 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, improve the stability and utilization of exosomes, but also have the advantages of long-term 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, it is urgent 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 suture containing mesenchymal stem cell exosomes, 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 bottles, 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.

[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-collagen suture: dissolve norepinephrine NE in Tris buffer, then add collagen COL 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-COL suture;

[0014] (3) Preparation of exosome-loaded norepinephrine-collagen suture: Sterile pNE-COL suture was placed in a certain concentration of exosome solution and placed in a 37°C constant temperature incubator for 1 hour to adsorb exosomes; then the exosome-absorbed suture was placed in a vacuum freeze dryer and vacuum freeze-dried at -75°C for 48 hours to obtain pNE-COL-Exo suture. The concentration of the exosome solution was 2.5-4.5 mg / mL.

[0015] The mesenchymal stem cell exosome-loaded absorbable suture prepared by this 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 collagen sutures, and through functional modification of the suture surface, umbilical cord mesenchymal stem cell exosomes are loaded onto the collagen sutures 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 collagen 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 COL-Exo suture and pNE-COL-Exo suture;

[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.13 Ki67 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 6The 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 method 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 future 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 980uL DPBS 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 can be confirmed that the obtained component is Hu-MSC-Exo.

[0050] 2. Preparation of pNE-COL-Exo Suture

[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 complete dissolution, COL 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 COL suture, i.e., pNE-COL suture, which was sterilized with ethylene oxide for later use.

[0053] The sterile pNE-COL 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-COL suture, i.e., pNE-COL-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-COL-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-COL-Exo sutures were tested. The test was divided into a pNE-COL-Exo suture group and a COL suture group. The COL 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 COL sutures, the breaking strength and breaking elongation of pNE-COL-Exo sutures were improved. This may be because 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] In order to observe whether exosomes were adsorbed to the surface of pNE-COL-Exo sutures, a cold field emission scanning electron microscope (Hitachi, Regulus8100) was used to observe the surface state of the sutures, and COL-Exo sutures were used as a control.

[0065] The results are as follows Figure 4 As shown, compared with COL sutures, pNE-COL 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 COL-Exo sutures and pNE-COL-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 5As 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-COL-Exo suture was significantly higher than that of the COL-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 fibroblasts by different sutures

[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] COL sutures, COL-Exo sutures, pNE-COL sutures, and pNE-COL-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 COL 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-COL-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. COL sutures, COL-Exo sutures, pNE-COL sutures, and pNE-COL-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-COL-Exo suture group has the strongest inhibitory ability on T lymphocytes, with an inhibition rate of 62.53%, while the COL suture group has the worst inhibitory ability on T lymphocytes, with an inhibition rate of 41.26%. This shows that the COL suture modified with pNE coating and adsorbed with Hu-MSC-Exo can significantly inhibit the proliferation of T lymphocytes and reduce local inflammatory response.

[0078] 6. Inhibitory effects 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-COL-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, COL sutures, COL-Exo sutures, pNE-COL sutures, and pNE-COL-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 COL suture group, COL-Exo suture group, pNE-COL suture group, and pNE-COL-Exo suture group were 9.91%, 2.33%, 3.84%, and 1.22%, respectively. Compared with the COL suture group, the sutures modified with pNE coating and Hu-MSC-Exo can significantly inhibit the proliferation of Th1 cells, and the pNE-COL-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-COL-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.

[0086] All sutures are sterilized with ethylene oxide before use, and all surgical instruments are sterilized with high temperature and high pressure before use.

[0087] 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 COL sutures and pNE-COL-Exo sutures, respectively, and a repair comparison experiment was performed.

[0088] 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.

[0089] 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-COL-Exo suture group was better, especially on the 7th day, there were fewer scars and some new hair.

[0090] 2. H&E staining of postoperative wound tissue

[0091] H&E staining was used to analyze the inflammatory response near the wound on the 3rd and 7th days after surgery.

[0092] The results are as follows Fig.10As shown, whether it is 3 days or 7 days after surgery, the epidermis of the wound skin tissue in the pNE-COL-Exo suture group is intact, the structure of each layer is clear, the dermis is thick, the number of skin appendages, hair follicles and sebaceous glands is abundant, the structure is clear, and the distribution is scattered. Fibroblast proliferation and new blood vessels can be seen locally; compared with the pNE-COL-Exo suture group, the epidermis of the skin tissue in the COL suture group is relatively intact, with a clear structure, but uneven thickness, more vacuolation of the spinous layer cells, more lymphocyte and granulocyte infiltration, more muscle cell necrosis, nuclear condensation, and increased cytoplasmic eosinophilia. The results show that pNE-COL-Exo has a significant promoting effect on early wound healing and can significantly improve the inflammatory response during wound healing.

[0093] 3. Collagen regeneration in postoperative wounds

[0094] 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).

[0095] 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 content of fibrous connective tissue in the subcutaneous injury site of the COL suture group decreased, with a small amount of collagen regeneration, and the positive area ratio was 29.3213±9.8127%, while the pNE-COL-Exo suture group had no pathological fibrosis and a large amount of collagen production, with a positive area ratio of 44.6640±3.7898%, which was 1.52 times that of the COL 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-COL-Exo suture group has more complete skin appendages, indicating that the pNE-COL-Exo suture has a better ability to promote tissue repair.

[0096] Table 2 Positive area ratio of two groups of sutures

[0097]

[0098] 4. Postoperative Angiogenesis

[0099] 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.

[0100] To verify the angiogenesis-promoting ability of pNE-COL-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 in the figure, although the positive expression of CD31 in the COL suture group increased from day 3 to day 7, it was only weakly positive (+). Compared with the COL suture group, the pNE-COL-Exo suture group had a stronger ability to promote angiogenesis, especially on day 7, the epidermis, sebaceous glands and hair cysts in the dermis, and smooth muscle of the muscular layer of the pNE-COL-Exo suture group were moderately strongly positively expressed (++), indicating that the pNE-COL-Exo suture has a stronger synergistic effect with angiogenesis and can significantly promote angiogenesis during wound healing.

[0101] 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%.

[0102] 5. Postoperative wound cell proliferation

[0103] 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.

[0104] 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-COL-Exo suture group and the COL suture group were weakly positive (+); on the 7th day, the epidermis, sebaceous glands in the dermis and smooth muscle of the muscular layer of the pNE-COL-Exo suture group were moderately positive (++), while the COL suture group was still weakly positive (+). This shows that COL sutures can also promote cell proliferation, but it is relatively slow, while pNE-COL-Exo sutures can quickly promote cell proliferation in the early stage of wound healing.

[0105] 6. Expression of macrophages in postoperative wounds

[0106] 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. Whether on the 3rd or 7th day, the pNE-COL-Exo suture group showed weak positive expression in the cytoplasm of the dermis and subcutaneous tissue, while the COL suture group showed moderate positive expression in the cytoplasm of the dermis and subcutaneous tissue (++). This shows that the pNE-COL-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 suture containing mesenchymal stem cell exosomes, 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 bottles, 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-collagen suture: dissolve norepinephrine NE in Tris buffer, then add collagen COL 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-COL suture; (3) Preparation of exosome-loaded norepinephrine-collagen sutures: Sterile pNE-COL sutures were placed in an exosome solution of a certain concentration and placed 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-COL-Exo sutures.

2. The method for preparing the absorbable suture containing mesenchymal stem cell exosomes 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, 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.

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

4. Use of the absorbable suture containing mesenchymal stem cell exosomes prepared by the method as claimed in claim 1 in wound suturing.

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