再生新动脉,完全自体脱细胞 z-bo 2020-12-29 352

　　Background: Autologous blood vessels such as saphenous vein and internal mammary artery are the gold standard grafts as vascular prostheses. Unfortunately, due to previous bypass surgery or existing vascular disease, these blood vessels are not always available or not suitable for surgery. When autologous blood vessels are not available, other artificial face transplants are often used. ? Polyester and polytetrafluoroethylene (PTFE) are two synthetic materials that are widely used in large-diameter (≥6 mm) transplantation, such as arteriovenous access for hemodialysis and peripheral arterial bypass above the knee. Unfortunately, due to infection, thrombosis, or intimal hyperplasia, these synthetic materials fail when used in small diameter (<6 mm) grafts. In the past three decades, tissue engineered vascular grafts (tevgs) have been extensively studied to explore immunocompatibility, non-thrombotic, growable and remodelable grafts. Many TEVGs, such as cell-seeded synthetic grafts, collagen and fibrin-based blood vessels, cell self-assembled blood vessels, and biodegradable synthetic polymer scaffolds have been developed. However, immune response, thrombosis, intimal hyperplasia, low mechanical strength and complications management procedures limit its clinical application. For the first time, Sparks et al. proposed a new method for preparing TEVGs using in vivo tissue engineering technology. Simply put, a rod is inserted under the skin and after a period of time, granulation tissue is formed. The rod is then removed, leaving an autologous tissue catheter that can be used as a blood vessel graft. This kind of graft is easy to obtain and has no immunity to the host, but it fails due to the low burst strength and low patency rate. Campbell et al. inserted and turned the tissue sac with a silicone tube as a stick to ensure that the inner wall was covered with non-thrombotic mesothelial cells. Although progress has been made, it is not suitable for clinical use due to poor mechanical properties, thrombosis and intimal hyperplasia caused by complications and unsatisfactory patency rates. Niklason and Tedd et al. used human homologous or autologous cells to design decellularized extracellular matrix vascular grafts for chronic hemodialysis. These grafts are easy to prepare, immunologically compatible, suitable for cell migration and growth, and can be re-implanted with new arteries in a miniature pig model.

　　 Preparation of autologous tissue catheter: Teflon tube (outer diameter 3.9 mm) is cut into 6 cm long tube sections, and then immersed in 75% alcohol for disinfection. Minipigs weighing 25-30 kg (n=8, females) were injected intramuscularly with zolazepam (5 mg/kg), xylazine (2.25 mg/kg) and atropine (1 mg) before anesthesia. Induce sedation with isoflurane, intubate the trachea, ventilate the animal with a mixture of oxygen and nitrogen (1:2 v/v), use the following ventilator settings: pressure control mode: positive end expiratory pressure (PEEP) 4cm H2O; Peak inspiratory pressure is 16-18cm H2O; respiratory rate is 12-16 times/min; tidal volume is 10 ml/kg. Fentanyl (10μg/kg/h) was injected intravenously through ear vein catheter to induce general anesthesia. After anesthesia, the pig was placed on the animal experiment table to prepare abdominal skin and disinfected with 0.1% povidone-iodine three times. A small incision of about 3 cm was made with a scalpel in the horizontal direction, and then a longitudinal subcutaneous capsule was formed by blunt dissection, and a tube was inserted into the capsule. A total of 8 tubes were inserted into 4 small incisions for each pig. After 4 weeks, collect the surrounding tissue ducts, remove the ducts, remove the excess tissue, remove the formed tissue ducts, and then store them together with 1% penicillin and streptomycin in 4°C PBS for further processing. Decellularization of autologous tissue ducts: Wash the prepared tissue ducts with PBS for 3 times, and then incubate them in a solution of 8 mM CHAPS, 1 mM NaCl, 0.12 mM NaOH and 25 mM EDTA on a stirring plate at 37°C For 2 hours, repeat 5 times with fresh decellularization solution to decellularize. The inoculum was then washed 3 times for 10 minutes in PBS on a stir plate. After the decellularization process, the graft is stored in 4°C PBS along with 1% penicillin and streptomycin for further processing or testing.

　　DNA quantification: quantify the total amount of DNA in non-depolarized or decellularized grafts using a tissue DNA isolation kit. First, the 40 mg wet weight sample was digested with cell lysis buffer and proteinase K, and then the protein fraction was removed by the protein precipitation solution and centrifugation. To separate DNA, the supernatant was added with isoamyl alcohol and ethanol, then centrifuged and rehydrated with DNA rehydration solution. Finally, the total DNA was quantified at 260 nm using a spectrophotometer.

　　 The burst pressure is measured by a flow system with a pressure sensor designed by the customer. Connect a 5 cm long graft to the flow system, inject PBS into the flow system, and increase the pressure in 50 mmHg increments until the graft fails, usually due to pinhole leakage or rupture. Record the maximum pressure as the burst pressure. The purpose of this test is to determine the force required to pull the suture from the prosthesis or cause the prosthesis wall to fail. Covalently connected to heparin: Heparin is covalently connected to the acellular tissue duct through the cross-linking between the succinimide ester on heparin and the amino function on the collagen, and the succinimide ester is covalently connected to the acellular tissue duct through EDC and NHS. The carboxylic acid group (hep-cooh) is activated. In more detail, the heparinized solution consists of solution A and solution B, where solution A uses EDC (40 mg/ml) and sulfonic NHS (20 mg/ml) in MES buffer (0.05 M, ph=5.5) Prepare at room temperature for 12 hours. Solution B is prepared with sodium heparin (60 mg/ml) in MES buffer. Mix Solution A and Solution B at room temperature at a volume ratio of 1:1 for 30 minutes to prepare a fresh heparinized solution, adjust the pH to 7 with 1 M NaOH solution, and then sterilize with a 2 μm filter. The acellular graft was soaked in the freshly prepared heparinized solution and placed on a stirring plate at room temperature for 5 h. After heparinization, the graft was washed with PBS. Toluidine blue staining was used to qualitatively observe the grafts connected with heparin. In short, 0.0005% toluidine blue solution was prepared in 0.01M hydrochloric acid and 0.2% (w/v) sodium chloride, the heparin-connected graft was incubated in toluidine blue solution overnight, and the graft without heparin was cultured For the control. The transplant was blue-purple, indicating that heparin is related to the transplant. The heparin-linked graft was cut into 5 mm long fragments, weighed and dissolved in 5 ml chloroform/acetone (1:1 v/v ratio), the solution was centrifuged at 14000g for 10 minutes, the precipitate was preserved, and chloroform/ The precipitate was washed twice with acetone solution, and the organic solvent was evaporated at room temperature to retain the heparin particles extracted from the graft. Then, they were immersed in 8 ml of toluene blue/n-hexane (1:1 v/v ratio) solution for 6 hours. At the same time, a series of standard heparin of known concentration was mixed in 8 ml of toluene blue/n-hexane (1:1 v/v). v) Incubate in solution for 6 hours. Use a plate reader to measure the absorbance of the sample and standard heparin solution at 630 nm. The heparin-linked grafts were cultured in PBS, shaken in a 37°C water bath for 12 h, and then the above qualitative test was performed.

　　Implantation and transplantation of the pig model: From 3 days before the operation, aspirin (5 mg/kg) and clopidogrel (1 mg/kg) were taken once a day. Continue to take aspirin and clopidogrel every day until the graft is transplanted. After anesthesia using the above method, the pig was placed on the dorsal animal experiment platform, and a 10 cm line was drawn above the left common carotid artery (1.5 cm from the midline). Establish an ear-edge vein channel to ensure fluid injection. All surgeries use strict aseptic conditions and techniques. A scalpel is used to cut the incision skin along the marked line, and the subcutaneous tissue is carefully cut with an electric knife. The superficial muscle is bluntly cut to expose the mastoid. Along the gap between the clavicle mastoid and the trachea, find the carotid sheath, and carefully dissect the left common carotid artery from the surrounding tissues using blunt dissection. Before clamping the artery, heparin (100 IU/kg) was injected intravenously, and the artery was flushed with trinitroglycerin to avoid vasospasm. Clamp the left common carotid artery with a non-traumatic vascular clip (5.7cm), cut the artery about 5 cm in length between the two clips, use 6-0 plerene as the end-to-end common carotid artery anastomosis, and suture it intermittently , Implantation of tissue catheters (decellularization and heparinization). The animals were randomly divided into two groups, and the vascular prostheses were taken out after the animals were sacrificed 1 month (n=5) and 2 months (n=3) after implantation. Double ultrasound was used to check patency before surgery. Heparin (100 IU/kg) was injected intravenously, the proximal and distal arteries were clamped, and the implanted graft was removed from the left common carotid artery. The removed grafts were cross-divided and washed with PBS, then fixed with formalin or stored at -80°C for further testing.

　　 Histological analysis: The common carotid artery, decellularized tissue catheter and graft after implantation were fixed in formalin and paraffin embedding, and 5 μm thick sections were cut with a microtome. Histological analysis used hematoxylin-eosin staining, Mason's trichrome staining, and Verhoeff staining. In order to identify endothelial cells and smooth muscle cells, immunostaining was performed with anti-von willebrand factor and alpha smooth muscle actin.

　　Result: Preparation of autologous tissue catheter: A smooth and unmodified Teflon tube with an outer diameter of 3.9 mm was cut into a 6.0 cm length. Immerse them in a 75% alcohol solution for 30 minutes, and then insert the sterile tube into the subcutaneous pocket on the abdomen of the miniature pig. After 4 weeks of subcutaneous implantation, a thick, autologous tissue duct was formed around the duct. It is easy to harvest and has almost no adhesion. Carefully remove the excess tissue, remove the catheter, and leave an autologous, smooth, thick tissue catheter, which can be used as a blood vessel transplant.

　　 Decellularization and heparinization analysis: The tissue ducts before decellularization are composed of cells and collagen-rich extracellular matrix. The cells should be fibroblasts and no elastin is seen. After decellularization, cellular components are rarely observed, and the extracellular matrix is rich in collagen. DAPI staining shows that CHAPS cleaner can remove cellular components very well. There are significant differences in DNA content before and after decellularization. The average thickness of non-decellularized, decellularized and native arteries were 324.1μm±57.4μm, 525.7μm±119.8μm, 637.4μm±50.6μm, respectively. There was no statistical difference in the thickness of decellularized and native arteries. Heparin binds to the acellular tissue duct through the sulfo NHS/EDC. After a water bath at 37°C for 12 hours, the average content of heparin bound to the tissue catheter was 8.2±0.9μg/mg.

　　 Surgery: The diameter of the tissue catheter matches the diameter of the carotid artery, and it is well anastomosed with the common carotid artery. As an interventional transplant, the transplant has smooth blood flow and obvious pulse. One month and two months after implantation, the implant did not experience thrombosis and the inner membrane was intact. Five pigs were followed up with Doppler ultrasound for 1 month. The patency rate was 100% (5/5), and the average inner diameter was 3.43±0.05 mm. Three pigs underwent Doppler ultrasound follow-up 2 months later. The patency rate was 67% (2/3), and the average inner diameter was 2.32±0.14mm. At 1 month and 2 months follow-up, there was no significant difference in the inner diameter of the two groups.

　　 Post-implantation histological analysis: After implantation, the cells migrated to the graft wall. In the first month after implantation, little elastin was formed on the graft wall, but almost no remodeling. Two months after implantation, the number of cells in the graft wall increased, and more mature wavy elastin was also observed in the graft wall. The results of immunofluorescence showed that a fused endothelial monolayer and a large number of α-smooth muscle actin (α-SMA) positive cells were observed on the entire graft wall. Conclusion: Our acellular autologous extracellular matrix vascular grafts show good patency and good in situ cellularization and reconstruction in the mini-pig model, including relatively early endothelialization and smooth muscle cell populations and good machinery performance. Our grafts have sufficient durability and patency, can be used for small diameter artery reconstruction, especially in peripheral arterial surgery, and may provide multiple treatment options in cardiovascular surgery.