Blood vessel biological patch and preparation thereof

Abstract

The invention belongs to the technical field of biological materials, and particularly discloses a blood vessel biological patch and preparation thereof, the blood vessel biological patch has the maximum tensile elongation of 20.1-35.1%, the elastic deformation rate of 15.1-29.4%, the value of the elastic deformation rate accounting for the maximum tensile elongation is 54-90%, and the bursting strength is 38-55N. The blood vessel biological patch provided by the 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 invention relates to a blood vessel biological patch and its preparation. Background Art

[0002] Stroke, commonly known as stroke, is a group of syndromes of limited or global brain dysfunction caused by acute cerebral circulation disorders, including two major categories: ischemic and hemorrhagic stroke. Ischemic stroke is cerebral infarction, and hemorrhagic stroke includes cerebral hemorrhage and subarachnoid hemorrhage. Stroke is characterized by high morbidity, high disability rate, high mortality rate and high recurrence rate. Carotid endarterectomy (CEA) uses surgical means to reopen the lumen and prevent embolus detachment and thrombosis, thereby preventing the occurrence of stroke. It is regarded as the first choice for treating carotid artery stenosis.

[0003] It is generally believed that the use of a patch to repair arterial occlusion can significantly reduce and prevent it. Patches are divided into venous patches, synthetic materials, or bovine pericardial patches. The following problems exist in the use of a patch for carotid endarterectomy: (1) The increase in surgical time and difficulty may increase the risk to the patient; (2) There is a lack of ideal patch materials. The venous patch used may rupture if it is too thin, and synthetic materials have risks; (3) Bleeding may occur at the sutures 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 five key parameter ranges of the maximum tensile elongation, elastic deformation rate, single-line suture pulling force, the value of the elastic deformation rate to the maximum tensile elongation, and bursting strength of the vascular biological patch, the obtained vascular biological patch can effectively reduce the problem of suture bleeding after carotid endarterectomy.

[0005] In addition, the inventors found through animal experiments that at the 4th and 8th weeks after surgery, the local performance of the biological patch was inconsistent, and some parts were softer or harder, that is, the biological patch had unstable performance. The inventors found that by controlling the maximum tensile elongation and bursting strength, the stable performance of the biological patch can be effectively guaranteed.

[0006] In the present invention, the determination of the maximum tensile elongation and the single-line suture pulling force refers to the existing literature (Li Chongchong, Liu Li, Wang Shuo, et al. Comparison of mechanical properties of allogeneic and animal-derived patches [J]. Beijing Biomedical Engineering, 2021.). The elastic deformation rate is determined using conventional determination methods in the field. 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. 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 drawn, and the elastic deformation rate of the vascular biological patch is calculated based on the elastic deformation segment of the tensile curve. The test method for bursting strength: fix the edge of a vascular patch of an appropriate area, and use a hard spherical crown with a diameter of Φ5 mm to apply a force at a speed of 100 mm / min to the vascular patch of the area corresponding to the spherical crown surface.

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

[0008] Preferably, 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.

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

[0010] Preferably, 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.

[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 another aspect of the present invention, it relates to a method for preparing the above-mentioned vascular biological patch, comprising:

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

[0015] (2) Placed in Cr 3+The ion concentration is 0.0625 mol / dm 3 , immersion treatment in hydroxy chromium solution with OH / Cr of 0.5 and pH of 2-3.

[0016] Preferably, in step (1), the bovine pericardium after removing the cell components, phospholipids, non-structural proteins and immunogenic molecules is immersed in a 0.05-0.1% glutaraldehyde solution for 3-3.5 hours, and then immersed in a 1.4-3.5% glutaraldehyde solution for 3-3.5 hours.

[0017] Preferably, in step (2), Cr 3+ The ion concentration is 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 a second water bath oscillation is performed.

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

[0019] The second water bath shaking was carried out at 36-45°C.

[0020] The suturing of the vascular biological patch provided by the present invention with the cutting edge of the carotid artery can improve the local hemodynamics of the carotid artery by expanding the diameter of the blood vessel through patch angioplasty, thereby inhibiting local thrombosis and intimal hyperplasia and reducing the incidence of carotid endarterectomy.

[0021] The vascular biological patch provided by the present invention uses bovine pericardium as raw material, has excellent compliance and tissue compatibility, is used for vascular reconstruction and repair, can effectively reduce bleeding at sutures, and the obtained biological patch has stable mechanical properties, and is a more promising material choice in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The pictures of the surgical process of animal experiment 1, wherein A: pictures during the operation; B: air drying damage to endothelial cells during simulated carotid endarterectomy; C: right carotid artery patch closure.

[0023] Figure 2 The cross-section of the vascular segment closed by the patch was stained with hematoxylin-eosin (magnification: ×20), where A, B, C, D, and E respectively represent different feeding times after surgery on the side closed by the patch; C: The patch is clearly distinguished from the intrinsic tissue, and the neointima is obvious on the surface of the patch. DETAILED DESCRIPTION

[0024] The following is further described in detail through specific implementation methods:

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

[0026] In order to further expand the range of mechanical optional parameters, and further verify the correlation between the four key parameters of maximum tensile elongation, elastic deformation rate, elastic deformation rate as a percentage of 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%, an elastic deformation rate as a percentage of the maximum tensile elongation of 54-90%, a single-line suture pulling force of 18.4-33.7N, and a bursting strength of 38-55N. Animal experiments were used again to verify the correlation between the maximum tensile elongation and bursting strength and the unstable performance of the biological patch, as well as the correlation between the elastic deformation rate, the elastic deformation rate as a percentage of the maximum tensile elongation and bleeding at the suture. Clinical experiments have verified that the use of biological patches for carotid endarterectomy vascular repair has the expected safety and effectiveness. According to the boundary 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%, elastic deformation rate accounts for 66-90% of the maximum tensile elongation, single-line suture pulling force 20.4-33.7N, bursting strength 41-55N. According to the embodiment in which the carotid endarterectomy is successfully completed, the biological patch fits tightly with the anastomosis, and the bleeding at the anastomosis and surrounding tissues can also be timely and effectively controlled, the parameter range of the biological patch finally optimized is: maximum tensile elongation 20.1-33.9%, elastic deformation rate 15.1-29.4%, elastic deformation rate accounts for 66-89% of the maximum tensile elongation, single-line suture pulling force 20.8-33.7N, bursting strength 41-54N.

[0027] 1. 18 preferred embodiments of digestive surgery biological patches and thoracic surgery biological patches (the contents of this section can also be implemented with reference to the records of patent application documents with patent application numbers 202411035176.0 and 202411087898.0)

[0028] 1. Preparation method

[0029] (1) Six preferred embodiments of preparation methods for digestive surgery biological patches

[0030] Step 1: Preprocessing

[0031] ① Soak healthy bovine pericardial tissue pieces in hypotonic Hank's solution, rinse them several times and replace the hypotonic Hank's solution repeatedly to fully swell and break up the various cells in the tissue.

[0032] ② Rinse the tissue slices treated as above repeatedly with physiological saline, each time for 60-110 minutes, and change the physiological saline each time. The total number of rinses is based on the absence of visible cells, cell components and cell fragments under the microscope of the tissue slices, and quantitative determination of protein and nucleic acid is performed until no soluble protein and nucleic acid can be detected.

[0033] Table 1: Rinse time of physiological saline solution for preferred embodiment of digestive surgery biological patch

[0034]

[0035]

[0036] ③ Use the surfactant solution Tween 80 to remove phospholipids and non-structural proteins in the tissue slices as well as some immunogenic molecules such as hyaluronic acid, various chondroitin sulfates and mucopolysaccharides.

[0037] ④ Soak in a 0.5-1.4% glutaraldehyde solution for 3-3.5 hours.

[0038] Table 2: Glutaraldehyde solution concentration and immersion time of preferred embodiment of digestive surgery biological patch

[0039] Group Glutaraldehyde solution concentration and immersion time Digestive surgery biological patch preferred embodiment 7 0.8％，3.5h Digestive surgery biological patch preferred embodiment 8 0.9％，3h Digestive surgery biological patch preferred embodiment 11 1.0％，3.5h Digestive surgery biological patch preferred embodiment 12 1.1％，3h Digestive surgery biological patch preferred embodiment 15 1.4％，3.5h Digestive surgery biological patch preferred embodiment 16 0.5％，3.5h

[0040] (2) Chemical modification

[0041] The pretreated tissue material was placed in Cr 3+ The ion concentration is 0.0625 mol / dm 3 , OH / Cr is 0.5, pH value is 2-3 (preferably pH value is 2.5-2.7), and the first water bath oscillation is carried out at 37-40°C for 3.5-5h. The pH value of the material treatment solution is detected, and 10% NaHCO3 is used to increase the pH value by 0.3-0.5 units, and then the second water bath oscillation is carried out at 41-45°C for 60-120 minutes to obtain a single-layer biopatch with a thickness of about 1mm.

[0042] Table 3: Glutaraldehyde solution concentration and immersion time of preferred embodiment of digestive surgery biological patch

[0043]

[0044] (2) Preparation methods of 12 preferred embodiments of thoracic surgery biological patches

[0045] Step 1: Preprocessing

[0046] ① Immerse healthy bovine pericardial tissue slices in hypotonic Hank's solution, rinse and replace the hypotonic Hank's solution repeatedly to fully swell and break up various cells in the tissue, and remove the swollen and broken cell fragments, cell nuclei and organelles after decellularization.

[0047] ② Rinse the tissue slices treated as above repeatedly with physiological saline. Each rinse lasts for 90-150 minutes. Change the physiological saline each time. The total number of rinses is based on the absence of visible cells, cell components and cell fragments under the microscope of the tissue slices. Quantitative determination of protein and nucleic acid is performed until no soluble protein and nucleic acid can be detected.

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

[0049] Group Rinse time of each saline solution Preferred embodiment 1 of the thoracic surgery biological patch 90min Preferred embodiment 4 of the thoracic surgery biological patch 90min Preferred embodiment 5 of the thoracic surgery biological patch 90min Preferred embodiment 8 of the thoracic surgery biological patch 90min Preferred embodiment 9 of the thoracic surgery biological patch 90min Preferred embodiment 10 of the thoracic surgery biological patch 90min Preferred embodiment 11 of the thoracic surgery biological patch 90min Preferred embodiment 12 of the thoracic surgery biological patch 90min Preferred embodiment 13 of the thoracic surgery biological patch 90min Preferred embodiment 18 of the thoracic surgery biological patch 120min Preferred embodiment 23 of the thoracic surgery biological patch 150min Preferred embodiment 24 of the thoracic surgery biological patch 150min

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

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

[0052] Table 5: Glutaraldehyde solution concentration and immersion time of preferred embodiment of thoracic surgery biological patch

[0053] Group Glutaraldehyde solution concentration and immersion time Preferred embodiment 1 of the thoracic surgery biological patch 2.5％，3h Preferred embodiment 4 of the thoracic surgery biological patch 2.5％，3h Preferred embodiment 5 of the thoracic surgery biological patch 2.5％，3h Preferred embodiment 8 of the thoracic surgery biological patch 2.5％，3h Preferred embodiment 9 of the thoracic surgery biological patch 2.5％，3h Preferred embodiment 10 of the thoracic surgery biological patch 2.5％，3h Preferred embodiment 11 of the thoracic surgery biological patch 2.5％，3h Preferred embodiment 12 of the thoracic surgery biological patch 2.5％，3h Preferred embodiment 13 of the thoracic surgery biological patch 2.5％，3h Preferred embodiment 18 of the thoracic surgery biological patch 2.5％，3h Preferred embodiment 23 of the thoracic surgery biological patch 3.5％，3h Preferred embodiment 24 of the thoracic surgery biological patch 4％，3.5h

[0054] Step 2: Chemical modification

[0055] The pretreated tissue material was placed in Cr 3+ The ion concentration is 0.0625 mol / dm 3 The first water bath oscillation is carried out in a hydroxychromium solution with an OH / Cr ratio of 0.5 and a pH value of 2-3. The conditions are: oscillation in a water bath at 20-28°C for 2-4 hours. The pH value of the material treatment solution is detected and 10% NaHCO3 is used to increase the pH value by 0.3-0.6 units. The second water bath oscillation is carried out under the conditions of oscillation in a water bath at 32-40°C for 3-5 hours to obtain a biological patch in the form of a single layer with a thickness of about 1 mm.

[0056] Table 6: Glutaraldehyde solution concentration and immersion time of preferred embodiment of thoracic surgery biological patch

[0057]

[0058]

[0059] 2. Mechanical testing

[0060] (1) Detection method

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

[0062] A 4 cm long and 1 cm wide biological patch 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 drawn, and the elastic deformation rate of the biological patch was calculated based on the elastic deformation section of the tensile curve during the test.

[0063] (2) Parameter range

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

[0065]

[0066]

[0067]

[0068] Based on the above table, it can be concluded that the maximum tensile elongation of 18 preferred embodiments of digestive surgery biological patches and thoracic surgery biological patches is 25.6-51.9%, the single-line suture pulling force is 17.2-37.2N, the elastic deformation rate is 14.6-39.4%, and the elastic deformation rate accounts for 49-93% of the maximum tensile elongation.

[0069] 2. Pulsating Flow Experiment

[0070] The pulsating 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 pulsating flow conditions, and to 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 on a pulsating flow platform. Under standard physiological conditions, after pulsating for 90 days, the sutures of the biological patches were observed to see if they were 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 pulsating flow experiment. It was concluded that for the vascular biological patch used in the carotid artery, the single-line suture pulling force should be no less than 20.3N.

[0071] 3. Tensile test to determine the tensile elongation at 80-140 mmHg

[0072] A biological patch of about 4 cm in length, 1 cm in width and 1 mm in thickness was clamped on a tensile testing machine along the length direction, and a tensile load of 0.35-0.61 N (converted from 80-140 mmHg, 101325 Pa = 760 mmHg, the inner diameter of the carotid artery is about 7 mm, and the thickness of the carotid artery is about 1 mm) was applied at a speed of 100 mm / min to stretch the biological patch, and the tensile elongation of the biological patch was observed. Tensile elongation = (length after stretching - original length) / original length × 100%.

[0073] Table 8: Tensile elongation of the digestive surgery biological patch and the thoracic surgery biological patch obtained in the preferred embodiment at 80-140 mmHg

[0074]

[0075]

[0076] It can be concluded from the above table that the relative values ​​of the tensile elongation and the maximum tensile elongation of the biological patch at 80-140 mmHg are consistent. Based on the fact that the deformation range of physiological blood vessels during contraction and expansion is 10-15%, if the tensile elongation of the biological patch is not within 10-15% at 80-140 mmHg, it is considered that the maximum tensile elongation of the biological patch is too large or too small.

[0077] Digestive surgery preferred embodiments 7, 8, 12, 15-16, and thoracic surgery preferred embodiments 5, 8, 11, 13 and 23 meet the requirements, and the maximum tensile elongation of the biological patches obtained in these embodiments ranges from 26.3% to 37.5%. The biological patches of these embodiments were subjected to animal experiments.

[0078] IV. Animal Experiments 1

[0079] 1. Experimental Design

[0080] Carotid artery surgery was performed on experimental animals using a preferred embodiment in which the tensile elongation of the biological patch was 10-15% under 80-140 mmHg. The carotid artery cavity was punctured with a needle and air was passed through for 10 minutes to damage endothelial cells, simulating the process of carotid endarterectomy.

[0081] 2. Selection of Experimental Animals

[0082] The experimental animals were male New Zealand white rabbits, 50 in number, weighing 2-3 kg, provided by Beijing Junwei Experimental Animal Breeding Center.

[0083] The experimental animals were kept for one week before surgery to adapt to the environment and quarantine. After surgery, the animals were kept in the animal room. The temperature of the facility was within the range of 16-26℃, and each animal was kept in a single cage. The cages were cleaned every day. They were fed with standardized feed, free food intake, and free drinking water.

[0084] 3. Surgical plan

[0085] 3.1 Preoperative preparation and anesthesia

[0086] After anesthesia with 2% sodium phenobarbital 1 ml / kg injected intravenously through the ear vein, the dose was increased according to the rabbit's state of consciousness.

[0087] 3.2 Surgical procedures

[0088] A median incision of about 2 cm in length was made in the rabbit's neck to expose the right carotid artery. A microaneurysm clip (FT7247, Aesculap AG, Braun, Germany) was used to block the proximal and distal ends of the carotid artery. The arterial cavity was then punctured with a needle and air was introduced to dry for 10 min (200 mL / min) to damage the endothelial cells, simulating the process of carotid endarterectomy. A glass float flowmeter (LZB-3, 30-300 mL / min, Senlod, China) was used to control the air flow rate. Subsequently, the carotid artery was longitudinally cut and closed using the biological patches obtained in 10 embodiments (one biological patch was used for each experimental animal. Since 10 biological patches were obtained in each embodiment, 2 biological patches were randomly selected from each embodiment for animal experiments) to observe whether the biological patch and the carotid artery were well anastomosed, whether the resilience of the biological patch met the requirements, whether the biological patch was fixed and stable, whether there was bleeding at the nail hole and suture, and the incision was sutured to complete the operation. After anesthesia, the rabbit woke up naturally. After the operation, all experimental animals were transferred to a professional breeding center and fed with high-cholesterol feed. Figure 1 The pictures of the surgical process of animal experiment 1, wherein A: pictures during the operation; B: air drying damage to endothelial cells during simulated carotid endarterectomy; C: right carotid artery patch closure.

[0089] All experimental animals were divided into 5 groups according to the different feeding and sacrifice times after surgery. The time intervals were 1, 2, 3, 4, and 8 weeks after surgery. Rabbits in each group were sacrificed at each time point. The carotid artery and surrounding tissues of the experimental animals that reached the end point were taken to observe the integrity of the patch, defects and leakage of the patch, whether there were thrombi on the surface, and whether there were changes such as bleeding and necrosis in the surrounding tissues. The pathological results of the experimental animals that reached the end point were recorded.

[0090] 3.3 Postoperative Care

[0091] After the operation is successful, the experimental animals are sent back to the animal room for further observation and feeding after recovery. Food and water are provided regularly. The animal's condition should be closely monitored during the entire postoperative care period, and the experimental animals should be observed for pain symptoms and given appropriate analgesics. Within 24 hours after surgery, the animals should be closely monitored, and then observed and recorded every day.

[0092] 4. Experimental Results

[0093] (1) Experimental animals using the biological patches obtained by the digestive surgery biological patch preferred embodiments 7-8, 16, and the thoracic surgery biological patch preferred embodiments 8, 11, 13 experienced bleeding at the sutures. The presumed reason is that the elastic deformation rate of the biological patch, as well as the value of the elastic deformation rate as a percentage of the maximum tensile elongation, are related to the bleeding at the sutures. When the elastic deformation rate and / or the value of the elastic deformation rate as a percentage of the maximum tensile elongation are small, bleeding will occur at the sutures.

[0094] The elastic deformation rates of the preferred embodiment 8 of the thoracic surgery biological patch and the preferred embodiment 15 of the digestive surgery biological patch are similar, but bleeding at the sutures occurred in the preferred embodiment 8 of the thoracic surgery biological patch. It can be concluded that when the elastic deformation rate is 23.5-29.2% and the elastic deformation rate accounts for 77-88% of the maximum tensile elongation, the biological patch can effectively avoid the problem of bleeding at the sutures.

[0095] (2) The experimental animals using the biological patches obtained by the preferred embodiment 15 of the digestive surgery biological patch and the preferred embodiments 5 and 23 of the thoracic surgery biological patch all successfully completed carotid endarterectomy. The biological patch fits tightly with the anastomosis, and the bleeding at the anastomosis and surrounding tissues can be effectively controlled in a timely manner. The anesthesia and anastomosis of the carotid artery and the biological patch were smooth, and there was no bleeding at the anastomosis during the operation; the vital signs were normal; and no adverse events occurred.

[0096] The animals were generally in good condition during the survival and breeding period, with normal body temperature, diet, and excretion, good autonomous activities, and no obvious abnormal manifestations such as weight loss, fever, anorexia, and mania. They survived to the end successfully without complications such as bleeding, infection, anastomotic stenosis, rejection reaction, and organ failure.

[0097] After the experiment, the animals were fed a high-cholesterol diet to simulate patients with poorly controlled risk factors, because a high-cholesterol diet can accelerate the process of intimal hyperplasia. Figure 2 As shown, the HE staining results show that the biological patch can be clearly distinguished from the surrounding blood vessel wall, indicating that the biological patches obtained by the preferred embodiment 15 of the digestive surgery biological patch and the preferred embodiments 5 and 23 of the thoracic surgery biological patch are stable in the experimental animals.

[0098] The relevant parameter ranges of the biological patches obtained by the preferred embodiment 15 of the digestive surgery biological patch and the preferred embodiments 5 and 23 of the thoracic surgery biological patch are: maximum tensile elongation 30.7-33.2%, elastic deformation rate 23.5-29.2%, single-line suture pulling force 21.5-36.1N, and the elastic deformation rate accounts for 77-88% of the maximum tensile elongation.

[0099] (3) In addition to the bleeding problem at the sutures, animal experiments also found that at the 4th and 8th weeks after surgery, the biological patches obtained using the preferred embodiments 8 and 13 of the thoracic surgery biological patches and the preferred embodiments 8 and 12 of the digestive surgery biological patches had unstable performance problems. The local performance of the biological patches was inconsistent, and some areas were softer or harder.

[0100] Combining Table 7, it can be found that the maximum tensile elongation of the patches obtained in these four embodiments is relatively large. However, the maximum tensile elongation 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 similar, but only the preferred embodiment 12 of the digestive surgery biological patch has the problem of unstable performance. Therefore, it is speculated that the unstable performance of the biological patch may be due to the influence of other parameter factors besides the maximum tensile elongation that the inventor has 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.

[0101] The inventors further conducted mechanical tests on the biological patches obtained from 10 embodiments (specifically, preferred embodiments 7-8, 12, 15-16 of the digestive surgery biological patch, and preferred embodiments 5, 8, 11, 13, and 23 of the thoracic surgery biological patch) with a tensile elongation of 10-15% at 80-140 mmHg, and unexpectedly found that, compared with the biological patch obtained from preferred embodiment 15 of the digestive surgery biological patch, the biological patches obtained from preferred embodiment 8 of the digestive surgery biological patch and preferred embodiments 8 and 13 of the thoracic surgery biological patch had a lower bursting strength except for a larger maximum tensile elongation.

[0102] The bursting strength test method is as follows: fix the edge of a vascular patch of appropriate area, use a hard spherical crown with a diameter of Φ5 mm to apply a force at a speed of 100 mm / min to the vascular patch of the area corresponding to the spherical crown surface, and test the bursting strength. The specific experimental results are shown in Table 9.

[0103] Table 9: Bursting strength of 10 examples with tensile elongation of 10-15% at 80-140 mmHg

[0104] Serial number Group Bursting strength 1 Digestive surgery biological patch preferred embodiment 7 44±0.3N 2 Digestive surgery biological patch preferred embodiment 8 34±0.3N 3 Digestive surgery biological patch preferred embodiment 12 39±0.1N 4 Digestive surgery biological patch preferred embodiment 15 46±0.2N 5 Digestive surgery biological patch preferred embodiment 16 50±0.1N 6 Preferred embodiment 5 of the thoracic surgery biological patch 44±0.6N 7 Preferred embodiment 8 of the thoracic surgery biological patch 25±0.4N 8 Preferred embodiment 11 of the thoracic surgery biological patch 49±0.1N 9 Preferred embodiment 13 of the thoracic surgery biological patch 22±0.5N 10 Preferred embodiment 23 of the thoracic surgery biological patch 45±0.3N

[0105] It can be concluded from Table 9 that the bursting strength of the 10 embodiments with a tensile elongation of 10-15% at 80-140 mmHg is 22-50 N.

[0106] In summary, it can be preliminarily concluded that controlling the maximum tensile elongation and elastic deformation rate is helpful to stably control the mechanical parameters of the biological patch.

[0107] Combined with the bursting strength parameter, it can be concluded that the relevant parameter ranges of the biological patch obtained in the embodiments (preferred embodiment 15 of the digestive surgery biological patch, and preferred embodiments 5 and 23 of the thoracic surgery biological patch) without bleeding at the sutures and with stable mechanical properties are: maximum tensile elongation 30.7-33.2%, single-line suture pulling force 21.5-36.1N, elastic deformation rate 23.5-29.2%, elastic deformation rate accounting for 77-88% of the maximum tensile elongation, and bursting strength 44-46N.

[0108] 5. Preparation method optimization

[0109] Based on the conclusions drawn from animal experiment 1, the preparation method was further optimized on the basis of the preparation methods of preferred embodiment 15 of the digestive surgery biological patch and preferred embodiments 5 and 23 of the thoracic surgery biological patch.

[0110] 1. Main differences between the preparation methods of the preferred embodiment 15 of the digestive surgery biological patch and the preferred embodiments 5 and 23 of the thoracic surgery biological patch

[0111] Table 10: Main differences in the preparation methods of preferred embodiment 15 of the digestive surgery biological patch and preferred embodiments 5 and 23 of the thoracic surgery biological patch

[0112]

[0113] 2. Preparation method optimization (Examples 1-12)

[0114] Step 1: Preprocessing

[0115] ① Immerse healthy bovine pericardial tissue slices in hypotonic Hank's solution, rinse and replace the hypotonic Hank's solution repeatedly to fully swell and break up various cells in the tissue, and remove the swollen and broken cell fragments, cell nuclei and organelles after decellularization.

[0116] ② Rinse the tissue slices treated as above repeatedly with physiological saline. Each rinse lasts 70-150 minutes. Change the physiological saline each time. The total number of rinses is based on the absence of visible cells, cell components and cell fragments under the microscope. Quantitative determination of protein and nucleic acid is performed until no soluble protein and nucleic acid can be detected.

[0117] Table 11: Saline rinse time

[0118] Group Rinse time of each saline solution 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 Embodiment 11 150min Example 12 150min

[0119] ③ Use the surfactant Tween 80 solution to remove phospholipids and non-structural proteins in the tissue slices as well as some tissue matrix immunogenic molecules such as hyaluronic acid, various chondroitin sulfates and mucopolysaccharides.

[0120] ④ After the first immersion in a 0.05-0.1% glutaraldehyde solution for 3-3.5 hours, the second immersion in a 1.4-3.5% glutaraldehyde solution for 3-3.5 hours.

[0121] Table 12: Glutaraldehyde solution concentration and immersion time

[0122]

[0123]

[0124] Step 2: Chemical modification

[0125] The pretreated tissue material was placed in Cr 3+ The ion concentration is 0.0625 mol / dm 3 The first water bath oscillation is carried out in a hydroxychromium solution with an OH / Cr ratio of 0.5 and a pH value of 2-3. The conditions are: water bath oscillation at 24-37°C for 2-5h.

[0126] The pH value of the material treatment liquid is tested and increased by 0.3-0.6 pH units with 10% NaHCO3 for 2-4 times, with each increase of at least 0.1 pH. Then, a second water bath oscillation is performed at 36-45°C for 2-5 hours to obtain a single-layer vascular biological patch with a thickness of about 1 mm.

[0127] Table 13: Water bath shaking conditions

[0128]

[0129] 3. Mechanical testing

[0130] (1) Detection method

[0131] Ten biological patches were obtained for each example.

[0132] Based on the existing literature (Li Chongchong, Liu Li, Wang Shuo, et al. Comparison of mechanical properties of allogeneic and animal-derived patches [J]. Beijing Biomedical Engineering, 2021.), the maximum tensile elongation and single-line suture pulling force were determined.

[0133] A 4 cm long and 1 cm wide biological patch 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 drawn, and the elastic deformation rate of the biological patch was calculated based on the elastic deformation section of the tensile curve during the test.

[0134] Fix the edge of a vascular patch of appropriate area, and use a hard spherical crown with a diameter of Φ5mm to apply a force at a speed of 100mm / min to the vascular patch of the area corresponding to the spherical crown surface to test the bursting strength.

[0135] (2) Parameter range

[0136] Table 14: Mechanical parameters of Examples 1-12

[0137]

[0138] In summary, it can be seen that the biological patches obtained in Examples 1-12 have a maximum tensile elongation of 20.1-35.1%, an elastic deformation of 15.1-29.4%, an elastic deformation of 54-90% of the maximum tensile elongation, a single-line suture pulling force of 18.4-33.7N, and a bursting strength of 38-55N.

[0139] VI. Animal Experiment 2

[0140] 1. Experimental Design

[0141] Observe the bleeding at the sutures of the biological patch and the stability of the mechanical parameters of the biological patch.

[0142] 2. Selection of Experimental Animals

[0143] The experimental animals were male New Zealand white rabbits, 60 in number, weighing 2-3 kg, provided by Beijing Junwei Experimental Animal Breeding Center.

[0144] The experimental animals were kept for one week before surgery to adapt to the environment and quarantine. After surgery, the animals were kept in the animal room. The temperature of the facility was within the range of 16-26℃, and each animal was kept in a single cage. The cages were cleaned every day. They were fed with standardized feed, free food intake, and free drinking water.

[0145] 3. Surgical plan

[0146] Refer to Animal Experiment 1.

[0147] 4. Experimental Results

[0148] (1) Among the experimental animals, only the experimental animals using the biological patch obtained in Example 6 experienced bleeding at the sutures. The elastic deformation rate of the biological patch obtained in this example accounted for a relatively small value of the maximum tensile elongation (54%).

[0149] (2) The biological patch obtained in Example 10 was torn at the suture line, and the single-line suture pulling force (18.4 N) of the biological patch obtained in this example was relatively small. This is consistent with the experimental results of the second pulsating flow experiment.

[0150] (3) Excluding the experimental animals with tearing and bleeding, the unstable performance of the biological patches in Examples 2, 4 and 12 occurred at the 4th and 8th weeks after surgery. It can be seen that after optimizing the preparation method, the stability of the mechanical parameters of the biological patches was greatly improved. By comparing the mechanical parameters, it can be found that the maximum tensile elongation of the biological patches obtained in Examples 2, 4 and 12 is larger, and / or the bursting strength is smaller, which once again verifies that the value range of the maximum tensile elongation and the bursting strength will affect the stability of the mechanical parameters of the biological patches.

[0151] In summary, the probability of bleeding at the suture and unstable performance of the biological patch obtained by using Examples 1-12 is low. The relevant mechanical parameters of the biological patch obtained by Examples 1-12 are: maximum tensile elongation 20.1-35.1%, elastic deformation rate 15.1-29.4%, elastic deformation rate accounting for 54-90% of the maximum tensile elongation, single-line suture pulling force 18.4-33.7N, and bursting strength 38-55N.

[0152] According to the boundaries of the parameters, it is further preferred that when the maximum tensile elongation is 20.1-33.9%, the elastic deformation rate is 15.1-29.4%, the elastic deformation rate accounts for 66-90% of the maximum tensile elongation, the single-line suture pulling force is 20.4-33.7N, and the bursting strength is 41-55N, the biological patch can effectively prevent bleeding at the sutures, as well as the problem of unstable performance and tearing of the biological patch at the sutures.

[0153] According to embodiments 1, 3, 5, 7-9 and 11, the carotid endarterectomy was successfully completed, the biological patch was tightly fitted to the anastomosis, and the bleeding at the anastomosis and surrounding tissues could be promptly and effectively controlled. It is further preferred that the biological patch with a maximum tensile elongation of 20.1-33.9%, an elastic deformation rate of 15.1-29.4%, an elastic deformation rate accounting for 66-89% of the maximum tensile elongation, a single-line suture pulling force of 20.8-33.7N, and a bursting strength of 41-54N can more effectively prevent bleeding at the sutures, as well as the problem of unstable performance and tearing of the biological patch at the sutures.

[0154] VII. Clinical Trials

[0155] 1. Purpose of the experiment

[0156] The biological patches obtained in Examples 1, 3, 5, 7-9 and 11 were used to evaluate whether the "vascular biological patch" had the expected safety and effectiveness when applied to vascular repair.

[0157] 2. Test content

[0158] The safety and effectiveness of vascular biological patches in vascular repair were evaluated through randomized, controlled, multicenter, non-inferiority clinical trials.

[0159] 3. Subject Selection

[0160] (1) Selection criteria

[0161] ① Asymptomatic and preoperative imaging examinations showed that the carotid stenosis was ≥70% or symptomatic and the carotid stenosis was >50%, and the final diagnosis criterion before inclusion was CTA; ② Physical condition and vital signs met the requirements for surgery; ③ The included patients were willing to comply with the trial protocol and were able to have regular follow-up examinations; ④ The subjects understood and / or their guardians and voluntarily signed the informed consent.

[0162] (2) Exclusion criteria

[0163] ①Expected survival time is less than 1 year; ②Cannot tolerate anesthesia; ③Cannot complete head and neck vascular CTA; ④Have had a large-area stroke or myocardial infarction within 30 days; ⑤Disorders of coagulation function, contraindications to heparin and antiplatelet drugs; ⑥Recent history of gastrointestinal bleeding, difficult to treat with antiplatelet drugs; ⑦With a large intracranial aneurysm, which cannot be treated in advance or at the same time; ⑧Chronic complete occlusion without obvious symptoms of cerebral ischemia; ⑨History of intracranial hemorrhage (intracerebral parenchyma, subarachnoid hemorrhage, subdural or epidural hemorrhage) within 30 days before inclusion; ⑩Combined with severe liver, kidney, circulatory system and other diseases; Severe dementia or mental disorder that precludes outpatient follow-up; Participants who have participated in other clinical trials within 3 months or are currently participating in other clinical trials; The researchers believe that the patients are not suitable for enrollment due to other reasons.

[0164] (3) Criteria and procedures for stopping a trial / trial treatment

[0165] ① During the study, the subject's condition continues to deteriorate and there is a possibility of life-threatening events. According to the doctor's judgment, the clinical researcher should be stopped, that is, the clinical research of this case should be terminated; ② During the study, the subject develops certain comorbidities, complications or special physiological and pathological changes and is unsuitable or unable to cooperate with the continued study; ③ During the clinical research, the subject is unwilling to continue the clinical research and requests the attending physician to terminate the clinical research, and the clinical research of this case can be terminated.

[0166] (4) The expected overall duration of the clinical trial and the reasons for its determination

[0167] The duration of this trial is set at 16 months from the first case to the last case. The reason is that one year after the vascular repair surgery, the effectiveness and safety of the trial product can be clearly observed. Each center expects the duration of enrollment to be 4 months. In this process, the clinical trial is divided into two stages: one is to make a full summary of the subject 6 months after enrollment; the other is to complete the clinical follow-up 1 year or more after surgery.

[0168] (5) The expected duration of participation for each subject is 1 year

[0169] (6) The number of subjects required for the clinical trial is 140 (10 subjects for each biological patch obtained in each example), including 70 subjects in the experimental group and 70 subjects in the control group. The control group uses a vascular patch registered by AESCULAP ​​AG, which is made of polyurethane.

[0170] 4. Effectiveness evaluation method

[0171] (1) Main research indicators: For carotid artery stenosis 1 year or more after surgery, postoperative neck vascular CTA showed stenosis > 50%. When the subject is free of stenosis, the treatment is considered effective for the subject, and the effective rate of each group of subjects is calculated. (2) Secondary research indicators: The incidence of perioperative complications after vascular biological patch angioplasty, including rupture of pseudoaneurysm, new cranial nerve injury after surgery, postoperative myocardial infarction, postoperative heart failure, postoperative ischemic stroke, postoperative hemorrhagic stroke, incisional hematoma, TIA (transient ischemic stroke), and postoperative wound. (3) Evaluate, record and analyze effectiveness parameters through neck vascular CTA examination at Visit 1, Visit 3, Visit 4 and Visit 5. (4) Safety evaluation methods: ① Vital signs, laboratory indicators, neurological recovery status, and incidence of adverse events.

[0172] 5. Test process

[0173] Table 15: Test process

[0174]

[0175]

[0176] Note: 1. Hemoglobin, white blood cell count, platelet count; 2. Total cholesterol, triglycerides, high-density lipoprotein, low-density lipoprotein, fasting blood sugar;

[0177] 3. Prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), and prothrombin time international normalized ratio (PTINR).

[0178] 6. Clinical trial results

[0179] (1) A total of 140 subjects were enrolled (70 in the experimental group and 70 in the control group), and 8 subjects dropped out. Dropout refers to the number of subjects who did not complete the clinical trial.

[0180] (2) The biological patches obtained in Examples 1, 3, 5, 7-9 and 11 are applied to the reconstruction and repair of blood vessels, and their safety and effectiveness have been clinically confirmed.

[0181] (3) One year or more after surgery, no restenosis of the carotid artery occurred in the experimental group, and stenosis was defined as >50% as indicated by postoperative CTA of the neck vessels. It is believed that the use of biological patches is effective in the treatment of subjects undergoing carotid endarterectomy.

[0182] During the perioperative period after angioplasty, no statistically significant differences were found between the experimental group and the control group in postoperative complications (including rupture of pseudoaneurysm, new cranial nerve injury after surgery, postoperative myocardial infarction, postoperative heart failure, postoperative ischemic stroke, postoperative hemorrhagic stroke, incisional hematoma, transient cerebral ischemia), neurological recovery, neck vascular examination results, and brain tissue examination, indicating that the use of biological patch for the treatment of subjects undergoing carotid endarterectomy is safe.

[0183] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection 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, and the bursting strength is 38-55N.

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 single-line suture pulling force of the vascular biological patch is 20.4-33.7N.

4. 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.

5. The vascular biological patch according to claim 4, characterized in that: The vascular biological patch is prepared by denaturing bovine pericardium with glutaraldehyde.

6. The vascular biological patch according to claim 5, characterized in that: The vascular biological patch contains chromium ions.

7. A method for preparing a vascular biological patch, characterized in that: include: (1) soaking the bovine pericardium after removing the cell components, phospholipids, non-structural proteins and immunogenic molecules in a glutaraldehyde solution with a concentration of 0.05-0.1% and a glutaraldehyde solution with a concentration of 1.4-3.5% successively; (2) Placed in Cr 3+ The ion concentration is 0.0625 mol / dm 3 , immersion treatment in hydroxy chromium solution with OH / Cr of 0.5 and pH of 2-3.

8. The method according to claim 7, characterized in that In step (1), the bovine pericardium after removing the cell components, phospholipids, non-structural proteins and immunogenic molecules is immersed in a glutaraldehyde solution with a concentration of 0.05-0.1% for 3-3.5 hours, and then immersed in a glutaraldehyde solution with a concentration of 1.4-3.5% for 3-3.5 hours.

9. The method according to claim 7, characterized in that: In step (2), place Cr 3+ The ion concentration is 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 a second water bath oscillation is performed.

10. The method according to claim 9, characterized in that In step (2), place Cr 3+ The ion concentration is 0.0625 mol / dm 3 The temperature of the first water bath oscillation in the hydroxychromium solution with an OH / Cr ratio of 0.5 and a pH value of 2-3 is 24-37°C; The second water bath shaking was carried out at 36-45°C.

Images