A vascular biological patch and its preparation

Abstract

The present invention belongs to the technical field of biological materials, and specifically discloses a vascular biological patch and its preparation. The maximum tensile elongation rate of the vascular biological patch is 20.1 - 35.1%, the elastic deformation rate is 15.1 - 29.4%, the value of the elastic deformation rate accounting for the maximum tensile elongation rate is 54 - 90%, and the bursting strength is 38 - 55 N. The vascular biological patch provided by the present invention can effectively reduce bleeding at the suture after carotid endarterectomy and prevent the problem of unstable performance of the biological patch.

Description

Technical Field

[0001] The present invention relates to a vascular biological patch and its preparation. Background Art

[0002] Stroke, commonly known as apoplexy, is a group of syndromes of focal or global cerebral functional deficits caused by acute cerebral circulatory disorders, including two major categories: ischemic and hemorrhagic strokes. Ischemic stroke is cerebral infarction, and hemorrhagic stroke includes cerebral hemorrhage and subarachnoid hemorrhage. Stroke is characterized by high incidence, high disability rate, high mortality rate, and high recurrence rate. Carotid endarterectomy (CEA) can reopen the lumen surgically and prevent embolus shedding and thrombus formation, thereby preventing stroke, and is regarded as the primary choice for treating carotid stenosis.

[0003] Generally, it is considered that after repair with a patch, arterial occlusion is significantly reduced and can be prevented. The patches are divided into vein patches, synthetic materials, or bovine pericardium patches, etc. There are the following problems with using a patch for carotid endarterectomy: (1) The increase in operation time and difficulty may increase the risk to the patient; (2) There is a lack of an ideal patch material. The vein patch used may be too thin and rupture, and there are risks with synthetic materials; (3) Bleeding may occur at the suture of the patch, and the mechanical parameters need to be further optimized. (4) The endothelialization rate is slow. Summary of the Invention

[0004] Through research, the present invention finds that by controlling the ranges of five key parameters, namely the maximum tensile elongation rate, elastic deformation rate, single - line suture pulling force, the value of the elastic deformation rate accounting for the maximum tensile elongation rate, and the bursting strength of the vascular biological patch, the obtained vascular biological patch can effectively reduce the bleeding problem at the suture after carotid endarterectomy.

[0005] In addition, through animal experiments, the present invention finds that at the 4th week and the 8th week after surgery, the performance of the local area of the biological patch is inconsistent, showing a situation where the local area is softer or harder, that is, the biological patch has a problem of unstable performance. The inventor finds that by controlling the maximum tensile elongation rate and the bursting strength, the performance stability of the biological patch can be effectively ensured.

[0006] In the present invention, the determination of the maximum tensile elongation rate and the single - line suture pulling force refers to the existing literature (Li Chongchong, Liu Li, Wang Shuo, et al. Comparison of the mechanical properties of allogeneic and animal - derived patches [J]. Beijing Biomedical Engineering, 2021.). The determination of the elastic deformation rate uses the conventional determination methods in the art. The prepared vascular biological patch is cut into a length of 4 cm and a width of 1 cm. The vascular biological patch is clamped on a tensile testing machine along its length direction, and a tensile load is applied at a speed of 100 mm / min to stretch the vascular biological patch until it breaks. A tensile curve is plotted, and based on the elastic deformation section of the tensile curve, the elastic deformation rate of the vascular biological patch is calculated. The test method for the bursting strength: Fix the edge of an appropriate area of the vascular patch, and apply a force to the vascular patch corresponding to the area of the spherical crown surface with a hard spherical crown with a diameter of Φ5 mm at a speed of 100 mm / min.

[0007] As an aspect of the present invention, it relates to a vascular biological patch. The maximum tensile elongation rate of the vascular biological patch is 20.1 - 35.1%, the elastic deformation rate is 15.1 - 29.4%, the value of the elastic deformation rate accounting for the maximum tensile elongation rate is 54 - 90%, and the bursting strength is 38 - 55 N.

[0008] Preferably, the maximum tensile elongation rate of the vascular biological patch is 20.1 - 33.9%, the value of the elastic deformation rate accounting for the maximum tensile elongation rate is 66 - 90%, and the bursting strength is 41 - 55 N.

[0009] Preferably, the single - line suture pulling force of the vascular biological patch is 20.4 - 33.7 N.

[0010] Preferably, the maximum tensile elongation rate of the vascular biological patch is 20.1 - 33.9%, the elastic deformation rate is 15.1 - 29.4%, the value of the elastic deformation rate accounting for the maximum tensile elongation rate is 66 - 89%, the single - line suture pulling force is 20.8 - 33.7 N, and the bursting strength is 41 - 54 N.

[0011] Preferably, the vascular biological patch is prepared by denaturing bovine pericardium with glutaraldehyde.

[0012] Preferably, the vascular biological patch contains chromium ions.

[0013] As yet another aspect of the present invention, it relates to a method for preparing the above - mentioned vascular biological patch, including:

[0014] (1) Soak the bovine pericardium after removing cell components, phospholipids, non - structural proteins, and immunogenic molecules successively in a glutaraldehyde solution with a concentration of 0.05 - 0.1% and a glutaraldehyde solution with a concentration of 1.4 - 3.5%;

[0015] (2) Place it in Cr 3+The ion concentration is 0.0625 mol / dm 3 , and it is soaked and treated with a hydroxyl chromium solution with an OH / Cr of 0.5 and a pH value of 2 - 3.

[0016] Preferably, in step (1), after taking out the cell components, phospholipids, non-structural proteins, and immunogenic molecules, the bovine pericardium is soaked in a glutaraldehyde solution with a concentration of 0.05 - 0.1% for 3 - 3.5 h, and then soaked in a glutaraldehyde solution with a concentration of 1.4 - 3.5% for 3 - 3.5 h.

[0017] Preferably, in step (2), it is placed in Cr 3+ The ion concentration is 0.0625 mol / dm 3 , and after the first water bath oscillation in a hydroxyl chromium solution with an OH / Cr of 0.5 and a pH value of 2 - 3, the pH value is increased by 0.3 - 0.6, and then the second water bath oscillation is carried out.

[0018] Preferably, in step (2), when placed in Cr 3+ The ion concentration is 0.0625 mol / dm 3 , and the temperature for the first water bath oscillation in a hydroxyl chromium solution with an OH / Cr of 0.5 and a pH value of 2 - 3 is 24 - 37°C;

[0019] The second water bath oscillation is carried out under the condition of 36 - 45°C.

[0020] The suture of the vascular biological patch provided by the present invention with the carotid artery resection margin can expand the blood vessel diameter through patchplasty, improve the local hemodynamics of the carotid artery, thereby inhibit local thrombus formation and intimal hyperplasia, and reduce the incidence rate caused by carotid endarterectomy.

[0021] The vascular biological patch provided by the present invention uses bovine pericardium as the raw material, has excellent compliance and tissue compatibility, is used for vascular reconstruction and repair, can effectively reduce bleeding at the suture, and the obtained biological patch has stable mechanical properties, which is a promising material choice in the future. Brief Description of the Drawings

[0022] Figure 1 Pictures of the surgical process for Animal Experiment 1, where A: Picture during the operation; B: Air-dried damaged endothelial cells simulating the process of carotid endarterectomy; C: Right carotid artery patch closure.

[0023] Figure 2 Cross-section hematoxylin-eosin staining (magnification: ×20) of the blood vessel segment closed by the patch, where A, B, C, D, E: respectively represent different feeding times after the operation on the patch closure side; C: The patch is significantly distinguishable from the native tissue, and the neointima on the surface of the patch is obvious. Detailed implementation manners

[0024] The following is a further detailed description through specific implementation manners:

[0025] In the process of completing the present invention, the inventor first conducted relevant mechanical experiments and animal experiments on the biological patches obtained from the preferred embodiments of the digestive surgery biological patch and the thoracic surgery biological patch, and screened out the biological patches that can meet the requirements for vascular reconstruction and repair. Among them, in the animal experiments, the inventor found that the elastic deformation rate and the value of the elastic deformation rate accounting for the maximum tensile elongation rate would affect the bleeding problem at the suture. In addition to the bleeding problem at the suture, the inventor also found that there were problems with different local performances of the biological patch at the 4th week and the 8th week after the operation (that is, there were problems with unstable mechanical parameter performance, with some parts being too hard or too soft). Combining the mechanical parameters, the inventor unexpectedly found that the maximum tensile elongation rate and the bursting strength would affect the unstable performance of the biological patch. Finally, considering the bleeding problem at the suture and the unstable performance of the biological patch comprehensively, the mechanical parameters of the biological patch that can be applied to vascular reconstruction and repair are as follows: the maximum tensile elongation rate is 30.7 - 33.2%, the single - line suture pulling force is 21.5 - 36.1 N, the elastic deformation rate is 23.5 - 29.2%, the value of the elastic deformation rate accounting for the maximum tensile elongation rate is 77 - 88%, and the bursting strength is 44 - 46 N.

[0026] In order to further expand the range of selectable mechanical parameters and further verify the correlation between the four key parameters of maximum tensile elongation, elastic deformation rate, the value of elastic deformation rate accounting for the maximum tensile elongation, and bursting strength and bleeding at the suture of the biological patch and unstable performance, the inventor further optimized the preparation method and obtained a maximum tensile elongation of 20.1 - 35.1%, an elastic deformation rate of 15.1 - 29.4%, the value of elastic deformation rate accounting for the maximum tensile elongation of 54 - 90%, a single - line suture pulling force of 18.4 - 33.7 N, and a bursting strength of 38 - 55 N. Through animal experiments again, the correlation between the maximum tensile elongation and bursting strength and the unstable performance of the biological patch was verified, as well as the correlation between the elastic deformation rate, the value of elastic deformation rate accounting for the maximum tensile elongation and bleeding at the suture. Through clinical experiments, it was verified that the application of the biological patch in carotid endarterectomy vascular repair has expected safety and effectiveness. According to the boundaries of the parameters, the parameter range of the optimized biological patch is: maximum tensile elongation 20.1 - 33.9%, elastic deformation rate 15.1 - 29.4%, the value of elastic deformation rate accounting for the maximum tensile elongation 66 - 90%, single - line suture pulling force 20.4 - 33.7 N, bursting strength 41 - 55 N. According to the example where carotid endarterectomy was successfully completed, the biological patch was closely attached to the anastomosis, and bleeding at the anastomosis and surrounding tissues could be effectively controlled in a timely manner. Finally, the optimized parameter range of the biological patch is: maximum tensile elongation 20.1 - 33.9%, elastic deformation rate 15.1 - 29.4%, the value of elastic deformation rate accounting for the maximum tensile elongation 66 - 89%, single - line suture pulling force 20.8 - 33.7 N, bursting strength 41 - 54 N.

[0027] I. 18 preferred embodiments of the digestive surgery biological patch and the thoracic surgery biological patch (this part of the content can also be implemented with reference to the patent application documents with patent application numbers 202411035176.0 and 202411087898.0)

[0028] 1. Preparation method

[0029] (1) Preparation methods of 6 preferred embodiments of the digestive surgery biological patch

[0030] Step 1. Pretreatment

[0031] ① Immerse the healthy bovine pericardium tissue slices in hypotonic Hank's solution, and repeatedly replace the hypotonic Hank's solution through multiple rinses to fully swell and break various cells existing in the tissue.

[0032] ②Rinse the tissue sections after the above treatment repeatedly with normal saline for 60 - 110 minutes each time, changing the normal saline each time. The total number of rinsing times is based on the fact that no visible cells, cell components or cell debris can be seen under the microscope for the tissue sections, and protein and nucleic acid quantitative determinations are carried out until no soluble protein and nucleic acid can be detected.

[0033] Table 1: Rinsing time of normal saline for the preferred embodiments of the biological patch in digestive surgery

[0034]

[0035]

[0036] ③Use the surfactant solution Tween 80 to remove phospholipids, unstructured proteins and some tissue matrices such as immunogenic molecules like hyaluronic acid, various chondroitin sulfates and mucopolysaccharides in the tissue sections.

[0037] ④Immerse in a glutaraldehyde solution with a concentration of 0.5 - 1.4% for 3 - 3.5 hours.

[0038] Table 2: Concentration and immersion time of glutaraldehyde solution for the preferred embodiments of the biological patch in digestive surgery

[0039] Group Concentration and soaking time of glutaraldehyde solution Preferred Example 7 of Digestive Surgery Biological Patch 0.8％，3.5h Preferred Example 8 of Digestive Surgery Biological Patch 0.9％，3h Preferred Example 11 of Digestive Surgery Biological Patch 1.0％，3.5h Preferred Example 12 of Digestive Surgery Biological Patch 1.1％，3h Preferred Example 15 of Digestive Surgery Biological Patch 1.4％，3.5h Preferred Example 16 of Digestive Surgery Biological Patch 0.5％，3.5h

[0040] (2) Chemical modification

[0041] Place the pretreated tissue material in a hydroxy chromium solution with a Cr 3+ ion concentration of 0.0625 mol / dm 3 , OH / Cr of 0.5, and a pH value of 2 - 3 (preferably a pH value of 2.5 - 2.7) for the first water bath oscillation. The conditions are: 37 - 40 °C for 3.5 - 5 hours. Detect the pH value of the material treatment solution and increase it by 0.3 - 0.5 pH units with 10% NaHCO3, and then carry out the second water bath oscillation. The conditions are: water bath oscillation at 41 - 45 °C for 60 - 120 minutes to obtain a biological patch in the form of a single-layer sheet with a thickness of about 1 mm.

[0042] Table 3: Concentration and immersion time of glutaraldehyde solution for the preferred embodiments of the biological patch in digestive surgery

[0043]

[0044] (2) Preparation methods for 12 preferred embodiments of the biological patch in thoracic surgery

[0045] Step 1. Pretreatment

[0046] ① Immerse the healthy bovine pericardium tissue slices in hypotonic Hank's solution, and repeatedly replace the hypotonic Hank's solution through multiple rinses to fully swell and break various cells in the tissue, so as to remove the swollen and broken cell debris, cell nuclei, and cell organelles after decellularization treatment.

[0047] ② Repeatedly rinse the tissue slices after the above treatment with normal saline, with each rinse in normal saline lasting for 90 - 150 minutes and replacing the normal saline each time. The total number of rinses is based on the condition that no visible cells or cell components and cell debris can be seen under the microscope for the tissue slices, and protein and nucleic acid quantitative measurements are carried out until no soluble protein and nucleic acid can be detected.

[0048] Table 4: Rinsing time of normal saline for the preferred embodiment of the thoracic surgery biological patch

[0049] Group Time for each saline rinsing Preferred Example 1 of Thoracic Surgery Biological Patch 90min Preferred Example 4 of Thoracic Surgery Biological Patch 90min Preferred Example 5 of Thoracic Surgery Biological Patch 90min Preferred Example 8 of Thoracic Surgery Biological Patch 90min Preferred Example 9 of Thoracic Surgery Biological Patch 90min Preferred Example 10 of Thoracic Surgery Biological Patch 90min Preferred Example 11 of Thoracic Surgery Biological Patch 90min Preferred Example 12 of Thoracic Surgery Biological Patch 90min Preferred Example 13 of Thoracic Surgery Biological Patch 90min Preferred Example 18 of Thoracic Surgery Biological Patch 120min Preferred Example 23 of Thoracic Surgery Biological Patch 150min Preferred Example 24 of Thoracic Surgery Biological Patch 150min

[0050] ③ Use the surfactant solution Tween 80 to remove phospholipids, non-structural proteins, and some tissue matrices such as hyaluronic acid, various chondroitin sulfates, and mucopolysaccharides in the tissue slices, which are immunogenic molecules.

[0051] ④ Immerse in a 2.5 - 4% glutaraldehyde solution for 3 - 3.5 hours.

[0052] Table 5: Concentration of glutaraldehyde solution and soaking time for the preferred embodiment of the thoracic surgery biological patch

[0053] Group Concentration and soaking time of glutaraldehyde solution Preferred Example 1 of Thoracic Surgery Biological Patch 2.5％，3h Preferred Example 4 of Thoracic Surgery Biological Patch 2.5％，3h Preferred Example 5 of Thoracic Surgery Biological Patch 2.5％，3h Preferred Example 8 of Thoracic Surgery Biological Patch 2.5％，3h Preferred Example 9 of Thoracic Surgery Biological Patch 2.5％，3h Preferred Example 10 of Thoracic Surgery Biological Patch 2.5％，3h Preferred Example 11 of Thoracic Surgery Biological Patch 2.5％，3h Preferred Example 12 of Thoracic Surgery Biological Patch 2.5％，3h Preferred Example 13 of Thoracic Surgery Biological Patch 2.5％，3h Preferred Example 18 of Thoracic Surgery Biological Patch 2.5％，3h Preferred Example 23 of Thoracic Surgery Biological Patch 3.5％，3h Preferred Example 24 of Thoracic Surgery Biological Patch 4％，3.5h

[0054] Step 2: Chemical modification

[0055] Place the pretreated tissue material in a hydroxy chromium solution with a Cr 3+ ion concentration of 0.0625 mol / dm 3 , an OH / Cr of 0.5, and a pH value of 2 - 3 for the first water bath oscillation. The conditions are: water bath oscillation at 20 - 28°C for 2 - 4 hours, detect the pH value of the material treatment solution, and use 10% NaHCO3 to increase the pH value by 0.3 - 0.6 pH units, and then conduct the second water bath oscillation. The conditions are: water bath oscillation at 32 - 40°C for 3 - 5 hours to obtain a biological patch in the form of a single-layer sheet with a thickness of about 1 mm.

[0056] Table 6: Concentration of glutaraldehyde solution and soaking time for the preferred embodiment of the thoracic surgery biological patch

[0057]

[0058]

[0059] 2. Mechanical testing

[0060] (1) Detection method

[0061] Based on existing literature (Li Chongchong, Liu Li, Wang Shuo, et al. Comparison of the mechanical properties of allogeneic and animal-derived patches [J]. Beijing Biomedical Engineering, 2021.), the maximum tensile elongation and single suture traction force were measured.

[0062] A biological patch with a length of 4 cm and a width of 1 cm was clamped on a tensile testing machine along the length direction, and a tensile load was applied at a speed of 100 mm / min to stretch the biological patch until it broke. A tensile curve was plotted, and based on the elastic deformation section of the tensile curve during the test, the elastic deformation rate of the biological patch was calculated.

[0063] (2) Parameter range

[0064] Table 7: Mechanical parameters of the preferred embodiments of the digestive surgery biological patch and the thoracic surgery biological patch

[0065]

[0066]

[0067] From the above table, it can be concluded that for the 18 preferred embodiments of the digestive surgery biological patch and the thoracic surgery biological patch, the maximum tensile elongation is 25.6 - 51.9%, the single suture traction force is 17.2 - 37.2 N, the elastic deformation rate is 14.6 - 39.4%, and the value of the elastic deformation rate accounting for the maximum tensile elongation is 49 - 93%.

[0068] II. Pulsatile flow experiment

[0069] The pulsatile flow experiment was used to evaluate the shear resistance of the biological patches obtained from 18 preferred embodiments of the digestive surgery biological patch and the thoracic surgery biological patch under pulsatile flow conditions, and preliminarily determine whether the biological patches obtained from the preferred embodiments can meet the application scenario requirements of the carotid artery. Specifically, according to the ISO 5840 standard, a biological patch with a length of 4 cm and a width of 1 cm was sewn onto the pulsatile flow platform. After 90 days of pulsation under standard physiological conditions, it was observed whether the suture of the biological patch was torn. The test results showed that only the biological patch obtained from the preferred embodiment 1 of the thoracic surgery biological patch was torn during the pulsatile flow experiment. From this, it can be concluded that for the vascular biological patch used in the carotid artery, the single suture traction force should be not less than 20.3 N.

[0070] III. Tensile experiment to measure the tensile elongation at 80 - 140 mmHg

[0071] A biological patch with a length of 4 cm, a width of 1 cm, and a thickness of about 1 mm is clamped on a tensile testing machine along the length direction, and a tensile load is applied at a speed of 100 mm / min with a tensile force of 0.35 - 0.61 N (obtained by converting 80 - 140 mmHg, 101325 Pa = 760 mmHg, the inner diameter of the carotid artery is about 7 mm, and the carotid artery thickness is about 1 mm) to stretch the biological patch, and the tensile elongation rate of the biological patch is observed. Tensile elongation rate = (length after stretching - original length) / original length × 100%.

[0072] Table 8: Tensile elongation rate of the patches obtained from the preferred embodiments of the digestive surgery biological patch and the thoracic surgery biological patch under 80 - 140 mmHg

[0073]

[0074]

[0075] It can be concluded from the above table that the relative numerical values of the tensile elongation rate and the maximum tensile elongation rate of the biological patch under 80 - 140 mmHg are the same. Based on the fact that the physiological blood vessel has a contraction and dilation deformation range of 10 - 15%, so if the tensile elongation rate of the biological patch is not within 10 - 15% under 80 - 140 mmHg, it is considered that the maximum tensile elongation rate of the biological patch is too large or too small.

[0076] Preferred embodiments 7, 8, 12, 15 - 16 of digestive surgery, and preferred embodiments 5, 8, 11, 13 and 23 of thoracic surgery meet the requirements, and the maximum tensile elongation rate range of the biological patches obtained from these embodiments is 26.3 - 37.5%. The biological patches of these embodiments are subjected to animal experiments.

[0077] IV. Animal Experiment 1

[0078] 1. Experimental design

[0079] Using the preferred embodiments with a tensile elongation rate of 10 - 15% of the biological patch under 80 - 140 mmHg to perform carotid artery surgery on experimental animals. The endothelial cells are damaged by puncturing the carotid artery lumen with a needle and introducing air to dry for 10 min to simulate the process of carotid endarterectomy.

[0080] 2. Selection of experimental animals

[0081] The experimental animals are 50 male New Zealand white rabbits, weighing 2 - 3 kg, provided by Beijing Junwei Experimental Animal Breeding Center.

[0082] The experimental animals were raised for one week before the operation to adapt to the environment and undergo quarantine. After the operation, the animals were raised in the animal house. The facility temperature was in the range of 16 - 26 °C, and each animal was raised individually in a cage. The cages were cleaned daily. Standardized feed was provided, and the animals had free access to food and water.

[0083] 3. Surgical procedure

[0084] 3.1 Preoperative preparation and anesthesia

[0085] After anesthesia by intravenous injection of 1 ml / kg of 2% sodium phenobarbital through the ear vein, the dose was increased according to the waking state of the rabbits.

[0086] 3.2 Surgical operation

[0087] Make an approximately 2-cm-long midline incision in the neck of the rabbit to expose the right carotid artery. Use a microaneurysm clip (FT7247, Aesculap AG, Braun, Germany) to block the proximal and distal ends of the carotid artery. Then, puncture the arterial lumen with a needle and introduce air to dry for 10 min (200 mL / min) to damage the endothelial cells, simulating the process of carotid endarterectomy. Use a glass rotameter (LZB-3, 30 - 300 mL / min, Senlod, China) to control the air flow rate. Subsequently, longitudinally incise the carotid artery and use the biological patches obtained from 10 examples (1 biological patch was used for each experimental animal, and since 10 biological patches were obtained from each example, 2 biological patches were randomly selected from each example for animal experiments) to close it. Observe whether the biological patch and the carotid artery are well anastomosed, whether the resilience of the biological patch meets the requirements, whether the biological patch is fixed stably, whether there is bleeding at the nail holes and suture sites, suture the incision to complete the operation, and the rabbit wakes up naturally after anesthesia. After the operation, all experimental animals were transferred to a professional breeding center and fed a high-cholesterol diet. Figure 1 Pictures of the surgical procedure for Animal Experiment 1, where A: Picture during the operation; B: Air-drying to damage endothelial cells during the process of simulating carotid endarterectomy; C: Patch closure of the right carotid artery.

[0088] All experimental animals were divided into 5 groups according to different postoperative feeding and sacrifice times. The time intervals were 1, 2, 3, 4, and 8 weeks after the operation. At each time point, the rabbits in each group were sacrificed. For the experimental animals that reached the end point, the carotid artery and surrounding tissues were taken, and gross observation was made to see if the patch was intact, had any defects or patch leakage, whether there was thrombosis on the surface, and whether there were changes such as bleeding and necrosis in the surrounding tissues. Record the pathological results of the experimental animals that reached the end point.

[0089] 3.3 Postoperative care

[0090] After the operation was successful, the experimental animals were revived and sent back to the animal house for continued observation and feeding. Food and water were provided regularly. During the entire postoperative care period, the animal status should be closely monitored to observe whether the experimental animals had pain symptoms, and analgesic drugs should be appropriately administered. Within 24 hours after the operation, the animals should be closely monitored, and then the animals should be observed every day to record their status.

[0091] 4. Experimental Results

[0092] (1) In the experimental animals using the biological patches obtained from Preferred Embodiment 7-8, 16 of the digestive surgery biological patch and Preferred Embodiment 8, 11, 13 of the thoracic surgery biological patch, bleeding occurred at the suture site. It is speculated that the reason is related to the elastic deformation rate of the biological patch, and the value of the elastic deformation rate accounting for the maximum tensile elongation rate and the bleeding at the suture site. When the elastic deformation rate and / or the value of the elastic deformation rate accounting for the maximum tensile elongation rate is small, bleeding will occur at the suture site.

[0093] The elastic deformation rates of Preferred Embodiment 8 of the thoracic surgery biological patch and Preferred Embodiment 15 of the digestive surgery biological patch are similar, but bleeding occurred at the suture site in Preferred Embodiment 8 of the thoracic surgery biological patch. Thus, it can be concluded that biological patches with an elastic deformation rate of 23.5 - 29.2% and a value of the elastic deformation rate accounting for the maximum tensile elongation rate of 77 - 88% can effectively avoid the problem of bleeding at the suture site.

[0094] (2) The experimental animals using the biological patches obtained from Preferred Embodiment 15 of the digestive surgery biological patch and Preferred Embodiment 5, 23 of the thoracic surgery biological patch all successfully completed carotid endarterectomy. The biological patch was closely adhered to the anastomosis, and the bleeding at the anastomosis and the surrounding tissues could be effectively controlled in a timely manner. The anesthesia during the operation, the anastomosis process of the carotid artery and the biological patch were smooth, and there was no bleeding at the intraoperative anastomosis; the vital signs were normal; no adverse events occurred.

[0095] During the survival and feeding period of the animals, the general condition was good, the body temperature, diet, and excretion were normal, the autonomous activities were good, and there were no obvious abnormal manifestations such as significant weight loss, fever, anorexia, mania, etc. They survived smoothly until the end point, and no complications such as bleeding, infection, anastomotic stenosis, rejection reaction, organ failure, etc. occurred in the surgical animals.

[0096] After the experiment, the experimental animals were fed a high-cholesterol diet to simulate patients with poor control of risk factors because a high-cholesterol diet can accelerate the process of intimal hyperplasia. As Figure 2 shown, the HE staining results showed that the biological patch could be clearly distinguished from the surrounding vascular wall, indicating that the biological patches obtained from Preferred Embodiment 15 of the digestive surgery biological patch and Preferred Embodiment 5, 23 of the thoracic surgery biological patch were stable in the experimental animals.

[0097] For the biological patch of the preferred embodiment 15 in the digestive surgery and the biological patches of the preferred embodiments 5 and 23 in the thoracic surgery, the relevant parameter ranges are as follows: the maximum tensile elongation rate is 30.7 - 33.2%, the elastic deformation rate is 23.5 - 29.2%, the single - line suture pulling force is 21.5 - 36.1 N, and the value of the elastic deformation rate accounting for the maximum tensile elongation rate is 77 - 88%.

[0098] (3) In addition to the bleeding problem at the suture site, animal experiments also found that in the 4th week and the 8th week after the operation, for the biological patches obtained from the preferred embodiments 8 and 13 of the thoracic - surgery biological patch and the preferred embodiments 8 and 12 of the digestive - surgery biological patch, there were problems of unstable performance, inconsistent local performance of the biological patch, and the situation of being locally softer or harder.

[0099] Combined with Table 7, it can be found that the maximum tensile elongation rates of the patches obtained from these 4 embodiments are relatively large. However, the maximum tensile elongation rates of the preferred embodiments 5 and 23 of the thoracic - surgery biological patch and the preferred embodiment 12 of the digestive - surgery biological patch are approximate. However, only the preferred embodiment 12 of the digestive - surgery biological patch has problems of unstable performance. Thus, it is speculated that there may be other parameter factors affecting the performance of the biological patch in addition to the maximum tensile elongation rate that the inventors have not discovered, making it impossible to stably control the mechanical parameters of the biological patch, which is not suitable for use as a surgical material.

[0100] The inventors further conducted further mechanical tests on the biological patches obtained from 10 embodiments (specifically, the preferred embodiments 7 - 8, 12, 15 - 16 of the digestive - surgery biological patch and the preferred embodiments 5, 8, 11, 13, 23 of the thoracic - surgery biological patch) where the tensile elongation rate of the biological patch is 10 - 15% under 80 - 140 mmHg. Surprisingly, compared with the biological patch obtained from the preferred embodiment 15 of the digestive - surgery biological patch, the biological patches obtained from the preferred embodiment 8 of the digestive - surgery biological patch and the preferred embodiments 8 and 13 of the thoracic - surgery biological patch have smaller bursting strengths in addition to larger maximum tensile elongation rates.

[0101] Test method for bursting strength: Fix the edge of the vascular patch with an appropriate area, and apply a force to the vascular patch corresponding to the area of the spherical crown with a diameter of Φ5 mm at a speed of 100 mm / min using a hard spherical crown facing the spherical crown, and test the bursting strength. For the specific experimental results, please refer to Table 9.

[0102] Table 9: Bursting strengths of 10 embodiments with a tensile elongation rate of 10 - 15% under 80 - 140 mmHg

[0103] Serial number Group Bursting strength 1 Preferred Example 7 of Digestive Surgery Biological Patch 44±0.3N 2 Preferred Example 8 of Digestive Surgery Biological Patch 34±0.3N 3 Preferred Example 12 of Digestive Surgery Biological Patch 39±0.1N 4 Preferred Example 15 of Digestive Surgery Biological Patch 46±0.2N 5 Preferred Example 16 of Digestive Surgery Biological Patch 50±0.1N 6 Preferred Example 5 of Thoracic Surgery Biological Patch 44±0.6N 7 Preferred Example 8 of Thoracic Surgery Biological Patch 25±0.4N 8 Preferred Example 11 of Thoracic Surgery Biological Patch 49±0.1N 9 Preferred Example 13 of Thoracic Surgery Biological Patch 22±0.5N 10 Preferred Example 23 of Thoracic Surgery Biological Patch 45±0.3N

[0104] It can be obtained from Table 9 that the bursting strength of 10 examples with a tensile elongation rate of 10 - 15% at 80 - 140 mmHg is 22 - 50 N.

[0105] In summary, it can be preliminarily concluded that controlling the maximum tensile elongation rate and elastic deformation rate helps to stably control the mechanical parameters of the biological patch.

[0106] Combined with the parameter of bursting strength, it can be obtained that the relevant parameter ranges of the biological patches obtained from the examples (Preferred Example 15 of the digestive surgery biological patch, and Preferred Examples 5 and 23 of the thoracic surgery biological patch) where there is no bleeding at the suture and the mechanical properties are stable are: the maximum tensile elongation rate is 30.7 - 33.2%, the single - suture pulling force is 21.5 - 36.1 N, the elastic deformation rate is 23.5 - 29.2%, the value of the elastic deformation rate accounting for the maximum tensile elongation rate is 77 - 88%, and the bursting strength is 44 - 46 N.

[0107] V. Optimization of the preparation method

[0108] Based on the conclusions drawn from Animal Experiment 1, on the basis of the preparation methods of Preferred Example 15 of the digestive surgery biological patch, and Preferred Examples 5 and 23 of the thoracic surgery biological patch, the preparation method is further optimized.

[0109] 1. Main differences in the preparation methods of Preferred Example 15 of the digestive surgery biological patch, and Preferred Examples 5 and 23 of the thoracic surgery biological patch

[0110] Table 10: Main differences in the preparation methods of Preferred Example 15 of the digestive surgery biological patch, and Preferred Examples 5 and 23 of the thoracic surgery biological patch

[0111]

[0112] 2. Optimization of the preparation method (Examples 1 - 12)

[0113] Step 1. Pretreatment

[0114] ① Immerse the healthy bovine pericardial tissue slices in hypotonic Hank's solution, and repeatedly rinse with the hypotonic Hank's solution replaced several times to fully swell and break various cells in the tissue, so as to remove the cell debris, cell nuclei and cell organelles that are swollen and broken after the decellularization treatment.

[0115] ② Repeatedly rinse the tissue slices treated above with physiological saline, each rinsing with physiological saline for 70 - 150 min, and replace the physiological saline each time. The total number of rinsing times is based on that no visible cells or cell components and cell debris can be seen under the microscope for the tissue slices, and protein and nucleic acid quantitative determination is carried out until no soluble protein and nucleic acid can be detected.

[0116] Table 11: Rinsing time with physiological saline

[0117] Group Time for each saline rinsing Example 1 70min Example 2 80min Example 3 90min Example 4 100min Example 5 110min Example 6 120min Example 7 130min Example 8 140min Example 9 150min Example 10 150min Example 11 150min Example 12 150min

[0118] ③ Use a surfactant Tween 80 solution to remove phospholipids, non-structural proteins, and some tissue matrices such as hyaluronic acid, various chondroitin sulfates, and mucopolysaccharides, which are immunogenic molecules, from the tissue sections.

[0119] ④ First, immerse in a 0.05 - 0.1% glutaraldehyde solution for 3 - 3.5 h, and then immerse in a 1.4 - 3.5% glutaraldehyde solution for 3 - 3.5 h for the second time.

[0120] Table 12: Glutaraldehyde solution concentration and immersion time

[0121]

[0122]

[0123] Step 2: Chemical modification

[0124] Place the pretreated tissue material in a hydroxy chromium solution with a Cr ion concentration of 0.0625 mol / dm, an OH / Cr of 0.5, and a pH value of 2 - 3 for the first water bath oscillation. The conditions are: water bath oscillation at 24 - 37 °C for 2 - 5 h. 3+ ion concentration of 0.0625mol / dm 3 and an OH / Cr of 0.5, and a pH value of 2 - 3 for the first water bath oscillation. The conditions are: water bath oscillation at 24 - 37 °C for 2 - 5 h.

[0125] Detect the pH value of the material treatment solution, and use 10% NaHCO3 to increase it by 0.3 - 0.6 pH units, for a total of 2 - 4 times, with each increase being at least 0.1 pH. Then, conduct the second water bath oscillation. The conditions are: water bath oscillation at 36 - 45 °C for 2 - 5 h to obtain a vascular biological patch in the form of a single-layer sheet with a thickness of about 1 mm.

[0126] Table 13: Water bath oscillation conditions

[0127]

[0128] 3. Mechanical testing

[0129] (1) Detection method

[0130] Ten biological patches are obtained for each example.

[0131] Based on existing literature (Li Chongchong, Liu Li, Wang Shuo, et al. Comparison of the mechanical properties of allogeneic and animal-derived patches [J]. Beijing Biomedical Engineering, 2021.), the maximum tensile elongation rate and single-line suture traction force are measured.

[0132] A 4-cm long and 1-cm wide biological patch is clamped on a tensile testing machine along the length direction, and a tensile load is applied at a speed of 100 mm / min to stretch the biological patch until it breaks. A tensile curve is plotted, and based on the elastic deformation section of the tensile curve during the test, the elastic deformation rate of the biological patch is calculated.

[0133] Fix the edge of a vascular patch with an appropriate area, and apply a force to the vascular patch corresponding to the area of the spherical crown surface with a hard spherical crown with a diameter of Φ5 mm at a speed of 100 mm / min to test the bursting strength.

[0134] (2) Parameter range

[0135] Table 14: Mechanical parameters of Examples 1-12

[0136]

[0137] In summary, it can be seen that the maximum tensile elongation rate of the biological patches obtained in Examples 1-12 is 20.1-35.1%, the elastic deformation rate is 15.1-29.4%, the value of the elastic deformation rate accounting for the maximum tensile elongation rate is 54-90%, the single-line suture pulling force is 18.4-33.7 N, and the bursting strength is 38-55 N.

[0138] VI. Animal experiment 2

[0139] 1. Experimental design

[0140] Observe the bleeding condition at the suture of the biological patch and the stability of the mechanical parameters of the biological patch.

[0141] 2. Selection of experimental animals

[0142] The experimental animals are 60 male New Zealand white rabbits, weighing 2-3 kg, provided by Beijing Junwei Experimental Animal Breeding Center.

[0143] One week before the experiment, the experimental animals are raised for environmental adaptation and quarantine. After the operation, the animals are raised in the animal house. The facility temperature is in the range of 16-26 °C, and each animal is raised in a single cage. The cage is cleaned every day. Standardized feed is fed, and the animals can eat freely and drink freely.

[0144] 3. Surgical plan

[0145] Refer to Animal experiment 1.

[0146] 4. Experimental results

[0147] (1) Among the experimental animals, only the experimental animals using the biological patch obtained in Example 6 showed bleeding at the suture, and the value of the elastic deformation rate of the biological patch obtained in this example accounting for the maximum tensile elongation rate (54%) is relatively small.

[0148] (2) The biological patch obtained in Example 10 had a tear at the suture, and the single - suture pulling force (18.4 N) of the biological patch obtained in this example was small. This is consistent with the experimental results of the second, pulsatile flow experiment.

[0149] (3) Excluding the experimental animals that had tears and bleeding, at the 4th and 8th weeks after surgery, Examples 2, 4, and 12 had problems with the instability of the biological patch performance. Thus, it can be seen that after optimizing the preparation method, the stability problem of the mechanical parameters of the biological patch has been greatly improved. By specifically comparing the mechanical parameters, it can be found that the biological patches obtained in Examples 2, 4, and 12 had a larger maximum tensile elongation rate and / or a smaller bursting strength, which again verified that the value ranges of the maximum tensile elongation rate and the bursting strength would affect the stability of the mechanical parameters of the biological patch.

[0150] In summary, the probability of the biological patches obtained by using Examples 1 - 12 having bleeding at the suture and instability of performance is low. The relevant mechanical parameters of the biological patches obtained by Examples 1 - 12 are: maximum tensile elongation rate 20.1 - 35.1%, elastic deformation rate 15.1 - 29.4%, the value of the elastic deformation rate accounting for the maximum tensile elongation rate 54 - 90%, single - suture pulling force 18.4 - 33.7 N, and bursting strength 38 - 55 N.

[0151] According to the boundaries of the parameters, further optimization shows that when the maximum tensile elongation rate is 20.1 - 33.9%, the elastic deformation rate is 15.1 - 29.4%, the value of the elastic deformation rate accounting for the maximum tensile elongation rate is 66 - 90%, the single - suture pulling force is 20.4 - 33.7 N, and the bursting strength is 41 - 55 N, the biological patch can effectively prevent bleeding at the suture and instability of performance, as well as the problem of the biological patch tearing at the suture.

[0152] According to Examples 1, 3, 5, 7 - 9, and 11 where carotid endarterectomy was successfully completed, the biological patch was closely adhered to the anastomosis, and bleeding at the anastomosis and the surrounding tissues could be effectively controlled in a timely manner. Further optimization shows that when the maximum tensile elongation rate is 20.1 - 33.9%, the elastic deformation rate is 15.1 - 29.4%, the value of the elastic deformation rate accounting for the maximum tensile elongation rate is 66 - 89%, the single - suture pulling force is 20.8 - 33.7 N, and the bursting strength is 41 - 54 N, the biological patch can more effectively prevent bleeding at the suture and instability of performance, as well as the problem of the biological patch tearing at the suture.

[0153] VII. Clinical Trials

[0154] 1. Test Purpose

[0155] Evaluate whether the "vascular biological patch" has the expected safety and effectiveness when applied to vascular repair by using the biological patches obtained in Examples 1, 3, 5, 7 - 9, and 11.

[0156] 2. Test content

[0157] Evaluate the safety and effectiveness of vascular biological patches in vascular repair through randomized, controlled, multi-center, non-inferiority clinical trials.

[0158] 3. Subject selection

[0159] (1) Inclusion criteria

[0160] ① Asymptomatic and preoperative imaging examination indicates carotid artery stenosis ≥ 70% or symptomatic and carotid artery stenosis > 50%. The final diagnosis criterion before inclusion is CTA; ② Physical condition and vital signs meet the surgical requirements; ③ The included patients are willing to comply with the trial protocol and have the ability to return for regular follow-up examinations; ④ The subjects understand and / or their guardians voluntarily sign the informed consent form.

[0161] (2) Exclusion criteria

[0162] ① Expected survival period less than 1 year; ② Unable to tolerate anesthesia; ③ Unable to complete head and neck vascular CTA; ④ Have had a large area of stroke or myocardial infarction within 30 days; ⑤ Coagulation dysfunction, contraindicated to heparin and antiplatelet drugs; ⑥ Have a recent history of gastrointestinal bleeding and it is difficult to carry out antiplatelet drug treatment; ⑦ Have a large intracranial aneurysm that cannot be treated in advance or simultaneously; ⑧ Chronic total occlusion without obvious cerebral ischemia symptoms; ⑨ Have a history of intracranial hemorrhage (intracerebral parenchyma, subarachnoid hemorrhage, subdural or epidural) within 30 days before inclusion; ⑩ Have severe liver, kidney, circulatory system and other diseases Severe dementia or mental disorder and unable to follow up in outpatient clinic; Have participated in other clinical trials within 3 months and currently; The researcher believes that there are other reasons not suitable for inclusion.

[0163] (3) Criteria and procedures for stopping the trial / trial treatment

[0164] ① During the study period, the subject's condition continues to deteriorate and there is a possibility of life-threatening events. According to the doctor's judgment, if it is necessary to stop the clinical research, the clinical research of this case will be terminated; ② During the research process, the subject has developed certain complications, comorbidities or special physiological and pathological changes, and is not suitable or unable to cooperate with the continuation of the research; ③ During the clinical research process, the subject is unwilling to continue the clinical research and requests to terminate the clinical research from the attending doctor, and the clinical research of this case can be terminated.

[0165] (4) Expected overall duration of the clinical trial and the reasons for its determination

[0166] This trial starts from the first case to the last case, and the duration is set at 16 months. The reason is that one year after vascular repair, the effectiveness and safety of the test product can be clearly observed. The expected enrollment duration for each center is 4 months. During this process, the clinical trial is divided into two phases. One is to conduct a full summary 6 months after the enrollment of the subjects. The other is to conclude the trial after 1 year or more of clinical follow-up after the operation.

[0167] (5) The expected participation duration for each subject is 1 year

[0168] (6) The number of subjects required for the clinical trial is 140 cases (10 subjects corresponding to the biological patch obtained in each example). Among them, there are 70 cases in the test group and 70 cases in the control group. The control group uses the vascular patch registered by AESCULAP AG of Snake Brand Co., Ltd., which is made of polyurethane.

[0169] 4. Methods for evaluating effectiveness

[0170] (1) Main research index: For the carotid artery 1 year and more after the operation, stenosis is defined as > 50% as indicated by CTA of the neck blood vessels after the operation. When there is no [specific situation not provided in the original] for the subject, it is considered that the treatment of the subject is effective, and the effective rate of each group of subjects is calculated. (2) Secondary research indexes: The incidence of complications during the perioperative period after vascular biological patch angioplasty, including rupture of pseudoaneurysm, newly occurred cranial nerve injury after the operation, myocardial infarction after the operation, heart failure after the operation, ischemic stroke after the operation, hemorrhagic stroke after the operation, incision hematoma, TIA (transient ischemic attack), and postoperative wound. (3) Through CTA examinations of the neck blood vessels at visit 1, visit 3, visit 4, and visit 5, evaluate, record, and analyze the effectiveness parameters. (4) Safety evaluation methods: ① Vital signs, laboratory indexes, neurological recovery status, and incidence of adverse events.

[0171] 5. Trial process

[0172] Table 15: Trial process

[0173]

[0174]

[0175] Note: 1. Hemoglobin, white blood cell count, platelet count; 2. Total cholesterol, triglyceride, high-density lipoprotein, low-density lipoprotein, fasting blood glucose;

[0176] 3. Prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), international normalized ratio of prothrombin time (PTINR).

[0177] 6. Clinical trial results

[0178] (1) A total of 140 cases were enrolled (70 cases in the experimental group and 70 cases in the control group), and 8 cases were lost to follow-up. Loss to follow-up refers to the number of subjects who did not complete the clinical trial.

[0179] (2) The biological patches obtained in Examples 1, 3, 5, 7-9 and 11 were applied to the reconstruction and repair of blood vessels, and their safety and effectiveness have been clinically confirmed.

[0180] (3) At 1 year or more after the operation, no restenosis of the carotid artery occurred in the experimental group. Restenosis was defined as >50% as indicated by CTA of the neck blood vessels after the operation. It is considered that the treatment of subjects undergoing carotid endarterectomy with the biological patch is effective.

[0181] During the perioperative period after vascular patch angioplasty, no statistically significant differences were found between the experimental group and the control group in postoperative complications (including rupture of pseudoaneurysm, new-onset cranial nerve injury after the operation, myocardial infarction after the operation, heart failure after the operation, ischemic stroke after the operation, hemorrhagic stroke after the operation, incision hematoma, transient ischemic attack), neurological recovery status, neck blood vessel examination results, and brain tissue examination. This indicates that the treatment of subjects undergoing carotid endarterectomy with the biological patch is safe.

[0182] The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those of ordinary skill in the art can make many modifications and variations based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by those skilled in the art in the technical field of the present invention based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the protection scope determined by the claims.

Claims

1. A vascular biological patch, characterized in that: The maximum tensile elongation of the vascular biological patch is 20.1-35.1%, the elastic deformation rate is 15.1-29.4%, the elastic deformation rate accounts for 54-90% of the maximum tensile elongation, the bursting strength is 38-55N, and the single-line suture pulling force is 20.4-33.7N; The method for preparing the vascular biological patch comprises: (1) Soak the bovine pericardium after removing the cell components, phospholipids, non-structural proteins and immunogenic molecules in a 0.05-0.1% glutaraldehyde solution for 3-3.5 hours, and then soak it in a 1.4-3.5% glutaraldehyde solution for 3-3.5 hours; (2) Place the Cr3+ ion concentration at 0.0625 mol / dm 3 After the first water bath oscillation in a hydroxychromium solution with an OH / Cr ratio of 0.5 and a pH value of 2-3, the pH value is increased by 0.3-0.6, and then a second water bath oscillation is performed; the temperature of the first water bath oscillation is 24-37°C; the second water bath oscillation is performed at 36-45°C.

2. The vascular biological patch according to claim 1, characterized in that: The maximum tensile elongation of the vascular biological patch is 20.1-33.9%, the elastic deformation rate accounts for 66-90% of the maximum tensile elongation, and the bursting strength is 41-55N.

3. The vascular biological patch according to claim 1, characterized in that: The maximum tensile elongation of the vascular biological patch is 20.1-33.9%, the elastic deformation rate is 15.1-29.4%, the elastic deformation rate accounts for 66-89% of the maximum tensile elongation, the single-line suture pulling force is 20.8-33.7N, and the bursting strength is 41-54N.

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