Vascular biopatch and preparation thereof

Abstract

The present invention belongs to the technical field of biomaterials, and particularly discloses a vascular biopatch and the preparation thereof. The vascular biopatch has a maximum tensile elongation of 20.1-35.1%, an elastic deformation rate of 15.1-29.4%, with the elastic deformation rate accounting for 54-90% of the maximum tensile elongation, and a burst strength of 38-55 N. The vascular biopatch provided by the present invention can effectively reduce bleeding at the suture after carotid endarterectomy and avoid the problem of unstable performance of the biopatch.

Description

A vascular biological patch and its preparation Technical Field

[0001] This invention relates to a vascular biological patch and its preparation. Background Technology

[0002] Stroke, commonly known as apoplexy, is a syndrome of localized or generalized brain dysfunction caused by acute cerebral circulatory disturbances. It is broadly classified into ischemic and hemorrhagic strokes. Ischemic stroke is cerebral infarction, while 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) is a surgical procedure that restores patency to the carotid artery and prevents emboli from dislodging and thrombus formation, thus preventing stroke. It is considered the primary treatment option for carotid artery stenosis.

[0003] It is generally believed that the use of patches for repair significantly reduces and prevents arterial occlusion. Patches are classified into venous patches, synthetic materials, or bovine pericardial patches. The following problems exist with using patches for carotid endarterectomy: (1) Increased surgical time and difficulty may increase patient risk; (2) There is a lack of ideal patch materials; thin venous patches may rupture, while synthetic materials pose risks; (3) Bleeding may occur at the suture site of the patch, and the mechanical parameters need further optimization; (4) Endothelialization is slow. Summary of the Invention

[0004] This invention has found that by controlling the range of five key parameters—maximum tensile elongation, elastic deformation rate, single-line suture traction force, elastic deformation rate as a percentage of maximum tensile elongation, and puncture strength—the resulting vascular biopatch can effectively reduce suture bleeding after carotid endarterectomy.

[0005] Furthermore, animal experiments conducted by this invention revealed inconsistencies in the local performance of the biological patch at weeks 4 and 8 post-surgery, with some areas exhibiting softer or harder textures, indicating performance instability. The inventors discovered that controlling the maximum tensile elongation and burst strength effectively ensures the performance stability of the biological patch.

[0006] In this invention, the determination of maximum tensile elongation and single-line suture traction force refers to 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 was determined using conventional methods in the field. The prepared vascular biopatch was cut to a length of 4 cm and a width of 1 cm. The vascular biopatch was clamped along its length on a tensile testing machine, and a tensile load was applied at a speed of 100 mm / min to stretch the vascular biopatch until it broke. A tensile curve was plotted, and the elastic deformation rate of the vascular biopatch was calculated based on the elastic deformation segment of the tensile curve. The burst strength test method: The edge of a vascular patch of appropriate area was fixed, and a rigid spherical cap with a diameter of Φ5 mm was used to apply a force to the vascular patch corresponding to the area of ​​the spherical cap at a speed of 100 mm / min.

[0007] As one aspect of the present invention, a vascular bio-patch is provided, wherein the vascular bio-patch 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 38-55 N.

[0008] Preferably, the vascular bio-patch has a maximum tensile elongation of 20.1-33.9%, an elastic deformation rate of 66-90% of the maximum tensile elongation, and a bursting strength of 41-55 N.

[0009] Preferably, the single-line suture traction force of the vascular biopatch is 20.4-33.7 N.

[0010] Preferably, the vascular bio-patch has 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 tensile force of 20.8-33.7N, and a bursting strength of 41-54N.

[0011] Preferably, the vascular biopatch is prepared from bovine pericardium by denaturation with glutaraldehyde.

[0012] Preferably, the vascular biopatch contains chromium ions.

[0013] As another aspect of the present invention, a method for preparing the above-mentioned vascular biological patch is provided, comprising:

[0014] (1) After removing cellular components, phospholipids, non-structural proteins and immunogenic molecules, the bovine pericardium was soaked in 0.05-0.1% glutaraldehyde solution and 1.4-3.5% glutaraldehyde solution, respectively.

[0015] (2) Placed in Cr 3+The ion concentration is 0.0625 mol / dm³. 3 Immersion treatment with a hydroxychromium solution with an OH / Cr ratio of 0.5 and a pH of 2-3.

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

[0017] Preferably, in step (2), the Cr is placed 3+ The ion concentration is 0.0625 mol / dm³. 3 After the first water bath shaking of a hydroxychromium solution with an OH / Cr ratio of 0.5 and a pH of 2-3, the pH is increased by 0.3-0.6, and then a second water bath shaking is performed.

[0018] Preferably, in step (2), the Cr is placed 3+ The ion concentration is 0.0625 mol / dm³. 3 The temperature for the first water bath shaking in a hydroxychromium solution with an OH / Cr ratio of 0.5 and a pH of 2-3 is 24-37℃.

[0019] The second water bath oscillation was carried out at 36-45℃.

[0020] The suturing of the vascular biological patch to the carotid artery resection margin provided by this invention expands the blood vessel diameter through patch formation, which can improve local hemodynamics of the carotid artery, thereby inhibiting local thrombosis and intimal hyperplasia, and reducing the incidence of carotid endarterectomy.

[0021] The vascular bio-patch provided by this invention uses bovine pericardium as raw material, has excellent compliance and tissue compatibility, and can be used for vascular reconstruction and repair. It can effectively reduce bleeding at the suture site, and the resulting bio-patch has stable mechanical properties, making it a promising material choice for the future. Attached Figure Description

[0022] Figure 1 shows the surgical procedure in animal experiment 1, where A: a picture during surgery; B: air drying damage to endothelial cells simulating carotid endarterectomy; C: right carotid artery patch closure.

[0023] Figure 2 shows a cross-section of the vascular segment closed by the patch, stained with hematoxylin and eosin (magnification: ×20). In the figure, A, B, C, D, and E represent different feeding times after the patch closure. In the figure, C shows a clear distinction between the patch and the intrinsic tissue, with obvious neointima on the patch surface. Detailed Implementation

[0024] The following detailed description illustrates the specific implementation method:

[0025] In developing this invention, the inventors first conducted relevant mechanical and animal experiments on biological patches obtained from preferred embodiments of digestive and thoracic surgical biological patches to screen for applications in vascular reconstruction and repair. In animal experiments, the inventors discovered that the elastic deformation rate and the value of the elastic deformation rate as a percentage of maximum tensile elongation affected suture site bleeding. Besides suture site bleeding, the inventors also observed localized performance variations in the biological patch at 4 and 8 weeks post-surgery (i.e., unstable mechanical parameters, with some areas being too hard or too soft). Based on these mechanical parameters, the inventors unexpectedly discovered that the maximum tensile elongation and burst strength affected the performance instability of the biological patch. Finally, considering the issues of bleeding at the suture site and the instability of the biological patch, the following mechanical parameters were selected for biological patches suitable for vascular reconstruction and repair: maximum tensile elongation 30.7-33.2%, single-suture traction force 21.5-36.1N, elastic deformation rate 23.5-29.2%, elastic deformation rate as a percentage of maximum tensile elongation 77-88%, and burst strength 44-46N.

[0026] To further expand the range of selectable mechanical parameters and to further verify the correlation between four key parameters—maximum tensile elongation, elastic deformation rate, the ratio of elastic deformation rate to maximum tensile elongation, and burst strength—and suture site bleeding and performance instability of the biological patch, the inventors further optimized the preparation method, obtaining a maximum tensile elongation of 20.1-35.1%, an elastic deformation rate of 15.1-29.4%, an elastic deformation rate to the ratio of maximum tensile elongation of 54-90%, a single-suture traction force of 18.4-33.7 N, and a burst strength of 38-55 N. Animal experiments again verified the correlation between maximum tensile elongation and burst strength and the performance instability of the biological patch, as well as the correlation between elastic deformation rate and the ratio of elastic deformation rate to maximum tensile elongation and suture site bleeding. Clinical trials verified the expected safety and efficacy of using the biological patch for vascular repair in carotid endarterectomy. Based on the parameter boundaries, the optimized parameter range for the biological patch is: maximum tensile elongation 20.1-33.9%, elastic deformation rate 15.1-29.4%, elastic deformation rate as a percentage of maximum tensile elongation 66-90%, single-suture traction force 20.4-33.7N, and burst strength 41-55N. Based on an example of successful carotid endarterectomy where the biological patch adhered tightly to the anastomosis, and bleeding at the anastomosis site and surrounding tissues was effectively controlled, the final optimized parameter range for the biological patch is: maximum tensile elongation 20.1-33.9%, elastic deformation rate 15.1-29.4%, elastic deformation rate as a percentage of maximum tensile elongation 66-89%, single-suture traction force 20.8-33.7N, and burst strength 41-54N.

[0027] I. Eighteen preferred embodiments of biological patches for digestive and thoracic surgery (This section can also be implemented with reference to the patent applications No. 202411035176.0 and No. 202411087898.0)

[0028] 1. Preparation method

[0029] (1) Preparation methods of six preferred embodiments of digestive surgical biological patches

[0030] Step 1: Preprocessing

[0031] ①Immerse healthy bovine pericardial tissue slices in hypotonic Hank's solution, and repeatedly rinse and replace the hypotonic Hank's solution to fully swell and break down the various cells present in the tissue.

[0032] ② Rinse the tissue slides treated above repeatedly with physiological saline for 60-110 minutes each time, changing the physiological saline each time. The total number of rinsing times depends on whether there are no morphological cells or cell components and cell debris under the microscope of the tissue slide. Perform quantitative determination of protein and nucleic acid until no soluble protein and nucleic acid can be detected.

[0033] Table 1: Saline rinsing time for preferred embodiments of digestive surgical biological patches

[0034] ③ Use the surfactant solution Tween 80 to remove phospholipids and non-structural proteins, as well as some tissue matrix molecules such as hyaluronic acid, various chondroitin sulfates, and mucopolysaccharides from the tissue slices.

[0035] ④ Soak in a 0.5-1.4% glutaraldehyde solution for 3-3.5 hours.

[0036] Table 2: Glutaraldehyde solution concentration and soaking time of preferred embodiments of digestive surgical biological patches

[0037] (2) Chemical modification

[0038] The pretreated tissue material was placed in Cr 3+ The ion concentration is 0.0625 mol / dm³. 3 The material was first treated with a hydroxychromium solution containing 0.5 OH / Cr and a pH of 2-3 (preferably 2.5-2.7) in a water bath at 37-40°C for 3.5-5 hours. The pH of the treatment solution was then measured and increased by 0.3-0.5 pH units with 10% NaHCO3. A second water bath was then performed at 41-45°C for 60-120 minutes to obtain a single-layer biological patch approximately 1 mm thick.

[0039] Table 3: Glutaraldehyde solution concentration and soaking time of preferred embodiments of digestive surgical biological patches

[0040] (2) Preparation methods of 12 preferred embodiments of thoracic surgical biological patches

[0041] Step 1: Preprocessing

[0042] ①Immerse healthy bovine pericardial tissue slices in hypotonic Hank's solution, and repeatedly rinse and replace the hypotonic Hank's solution to fully swell and break down various cells in the tissue, thereby removing cell fragments, nuclei and organelles that have been swollen and broken down after decellularization.

[0043] ② Rinse the tissue slides treated above repeatedly with physiological saline for 90-150 minutes each time, changing the physiological saline each time. The total number of rinsing times depends on whether there are no morphological cells or cell components and cell debris under the microscope of the tissue slide. Perform quantitative determination of protein and nucleic acid until no soluble protein and nucleic acid can be detected.

[0044] Table 4: Saline rinsing time for preferred embodiments of thoracic surgical biological patches

[0045] ③ Use the surfactant solution Tween 80 to remove phospholipids and non-structural proteins, as well as some tissue matrix molecules such as hyaluronic acid, various chondroitin sulfates, and mucopolysaccharides from the tissue slices.

[0046] ④ Soak in a 2.5-4% glutaraldehyde solution for 3-3.5 hours.

[0047] Table 5: Glutaraldehyde solution concentration and soaking time of preferred embodiments of thoracic surgical biological patches

[0048] Step 2, Chemical Modification

[0049] The pretreated tissue material was placed in Cr 3+ The ion concentration is 0.0625 mol / dm³. 3 The material was first shaken in a hydroxychromium solution with an OH / Cr ratio of 0.5 and a pH of 2-3 in a water bath at 20-28°C for 2-4 hours. The pH of the treatment solution was measured and increased by 0.3-0.6 pH units with 10% NaHCO3. Then, a second water bath shaking was performed at 32-40°C for 3-5 hours to obtain a single-layer sheet-like biological patch with a thickness of about 1 mm.

[0050] Table 6: Glutaraldehyde solution concentration and soaking time of preferred embodiments of thoracic surgical biological patches

[0051] 2. Mechanical Testing

[0052] (1) Detection method

[0053] Based on 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 traction force were determined.

[0054] A 4cm long and 1cm wide biological patch was clamped along its length onto a tensile testing machine. A tensile load was applied at a speed of 100mm / min to stretch the biological patch until it broke. A tensile curve was plotted, and the elastic deformation rate of the biological patch was calculated based on the elastic deformation segment of the tensile curve during the test.

[0055] (2) Parameter range

[0056] Table 7: Mechanical parameters of preferred embodiments of digestive surgery biological patches and thoracic surgery biological patches

[0057] Based on the table above, the maximum tensile elongation of the 18 preferred embodiments of the digestive surgery biological patch and the thoracic surgery biological patch is 25.6-51.9%, the single-line suture traction 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.

[0058] II. Pulsating Flow Experiment

[0059] The shear resistance of 18 preferred embodiments of digestive and thoracic surgical biological patches under pulsating flow conditions was evaluated using a pulsating flow test to preliminarily determine whether the biological patches obtained from the preferred embodiments could meet the requirements of carotid artery applications. Specifically, according to ISO 5840 standard, a biological patch measuring 4 cm in length and 1 cm in width was sewn onto a pulsating flow platform. After 90 days of pulsation under standard physiological conditions, the sutures of the biological patch were observed for tearing. The test results showed that only the biological patch obtained from preferred embodiment 1 of the thoracic surgical biological patch tore during the pulsating flow test. Therefore, it was concluded that for vascular biological patches used in the carotid artery, the single-suture traction force should not be less than 20.3 N.

[0060] III. Tensile Test: Determination of Elongation at 80-140 mmHg

[0061] A biological patch, approximately 4 cm long, 1 cm wide, and 1 mm thick, was clamped along its length onto a tensile testing machine. A tensile load of 0.35-0.61 N (calculated using 80-140 mmHg conversion; 101325 Pa = 760 mmHg; the inner diameter of the carotid artery is approximately 7 mm, and the thickness is approximately 1 mm) was applied at a speed of 100 mm / min to stretch the biological patch. The tensile elongation was observed. Tensile elongation = (Length after stretching - Original length) / Original length × 100%.

[0062] Table 8: Tensile elongation of bio-patches obtained from preferred embodiments of digestive and thoracic surgical bio-patches at 80-140 mmHg.

[0063] The table above shows that the relative values ​​of the tensile elongation and maximum tensile elongation of the biological patch at 80-140 mmHg are consistent. Based on the fact that physiological blood vessels deform within a range of 10-15% during contraction and expansion, if the tensile elongation of the biological patch is not within 10-15% at 80-140 mmHg, then the maximum tensile elongation of the biological patch is considered to be too large or too small.

[0064] Preferred embodiments 7, 8, 12, 15-16 for digestive surgery and preferred embodiments 5, 8, 11, 13 and 23 for thoracic surgery meet the requirements, and the maximum tensile elongation of the biological patches obtained by these embodiments ranges from 26.3% to 37.5%. Animal experiments were conducted on the biological patches of these embodiments.

[0065] IV. Animal Experiments 1

[0066] 1. Experimental Design

[0067] In a preferred embodiment where the elongation rate of the biological patch is 10-15% at 80-140 mmHg, carotid artery surgery was performed on experimental animals. The carotid artery was punctured with a needle and air was introduced for 10 minutes to damage the endothelial cells, simulating the process of carotid endarterectomy.

[0068] 2. Selection of laboratory animals

[0069] The experimental animals were 50 male New Zealand white rabbits, each weighing 2-3 kg, provided by Beijing Junwei Experimental Animal Breeding Center.

[0070] The experimental animals were kept in an acclimatization facility and quarantined for one week prior to surgery. Post-surgery, the animals were housed in the animal room. The facility temperature was maintained between 16-26℃, and each animal was kept in an individual cage. The cages were cleaned daily. Standardized feed was provided, and the animals had free access to food and water.

[0071] 3. Surgical plan

[0072] 3.1 Preoperative preparation and anesthesia

[0073] After anesthetizing the rabbit by intravenous injection of 1 ml / kg of 2% sodium phenobarbital via the ear vein, the dosage was increased according to the rabbit's level of consciousness.

[0074] 3.2 Surgical Procedure

[0075] A 2cm incision was made in the rabbit's neck to expose the right carotid artery. A miniature aneurysm clip (FT7247, Aesculap AG, Braun, Germany) was used to occlude the proximal and distal ends of the carotid artery. The arterial lumen was then punctured with a needle and air-dried for 10 minutes (200mL / min) to damage the endothelial cells, simulating a carotid endarterectomy. The airflow rate was controlled using a glass float flowmeter (LZB-3, 30-300mL / min, Senlod, China). The carotid artery was then longitudinally incised, and closed using a biological patch obtained from 10 examples (one patch per animal; since each example yielded 10 patches, two were randomly selected for animal experiments). The anastomosis between the biological patch and the carotid artery was observed, as well as the patch's resilience, stability, and the absence of bleeding at the pin holes and sutures. The incision was then sutured to complete the surgery, and the rabbit naturally awoke after anesthesia. After the surgery, all experimental animals were transferred to a specialized breeding center and fed a high-cholesterol diet. Figure 1 shows the surgical procedure in animal experiment 1, where A: a picture during the surgery; B: air-drying damage to endothelial cells simulating the carotid endarterectomy procedure; C: right carotid artery patch closure.

[0076] All experimental animals were divided into 5 groups based on their postoperative feeding and sacrifice time. The time intervals were 1, 2, 3, 4, and 8 weeks postoperatively. Rabbits in each group were sacrificed at each time point. The carotid artery and surrounding tissues of the experimental animals that reached the endpoint were harvested. The integrity of the patch, the presence of defects or leaks in the patch, the presence of thrombi on the surface, and the presence of hemorrhage, necrosis, or other changes in the surrounding tissues were observed. The pathological results of the experimental animals that reached the endpoint were recorded.

[0077] 3.3 Postoperative care

[0078] After successful surgery, the experimental animals were resuscitated and returned to the animal facility for continued observation and care. Food and water were provided regularly. Throughout the postoperative care period, the animals' condition was closely monitored, observing for signs of pain and administering appropriate analgesics. Close monitoring of the animals was necessary for the first 24 hours post-surgery, followed by daily observation and recording of their condition.

[0079] 4. Experimental Results

[0080] (1) Experimental animals using biological patches obtained from preferred embodiments 7-8 and 16 of digestive surgery biological patches and preferred embodiments 8, 11 and 13 of thoracic surgery biological patches experienced bleeding at the suture site. It is speculated that the elastic deformation rate of the biological patch and the value of the elastic deformation rate as a percentage of the maximum tensile elongation are related to bleeding at the suture site. 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 suture site.

[0081] The elastic deformation rates of the preferred embodiment 8 of the thoracic surgical biological patch and the preferred embodiment 15 of the digestive surgical biological patch are similar. However, the preferred embodiment 8 of the thoracic surgical biological patch experienced bleeding at the suture site. Therefore, it can be concluded that a biological patch with an elastic deformation rate of 23.5-29.2% and an elastic deformation rate accounting for 77-88% of the maximum tensile elongation can effectively avoid bleeding at the suture site.

[0082] (2) Experimental animals using the biological patches obtained in the preferred embodiment 15 of digestive surgery and the preferred embodiments 5 and 23 of thoracic surgery successfully underwent carotid endarterectomy. The biological patches adhered tightly to the anastomosis, and bleeding at the anastomosis site and surrounding tissues was effectively controlled in a timely manner. Intraoperative anesthesia and the anastomosis of the carotid artery with the biological patch were smooth, and there was no bleeding at the anastomosis site during the operation; vital signs were normal; and no adverse events occurred.

[0083] During the period of survival and rearing, the animals were generally in good condition, with normal body temperature, diet and excretion, good independent activity, and no obvious abnormalities such as weight loss, fever, anorexia, or mania. They survived to the end without complications such as bleeding, infection, anastomotic stenosis, rejection, or organ failure.

[0084] After the experiment, the experimental animals were fed a high-cholesterol diet to simulate patients with poorly controlled risk factors, as a high-cholesterol diet can accelerate the process of intimal hyperplasia. As shown in Figure 2, HE staining results showed that the biological patch was clearly distinguishable from the surrounding blood vessel wall, indicating that the biological patches obtained in preferred embodiment 15 of the digestive surgery biological patch and preferred embodiments 5 and 23 of the thoracic surgery biological patch were stable in the experimental animals.

[0085] The relevant parameter ranges of the biological patches obtained in preferred embodiment 15 of digestive surgery and preferred embodiments 5 and 23 of thoracic surgery are: maximum tensile elongation 30.7-33.2%, elastic deformation rate 23.5-29.2%, single-line suture traction force 21.5-36.1N, and the elastic deformation rate accounting for 77-88% of the maximum tensile elongation.

[0086] (3) In addition to bleeding at the suture site, animal experiments also found that the biological patches obtained by using 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 had unstable performance in the 4th and 8th weeks after surgery. The local performance of the biological patch was inconsistent, with some areas being softer or harder.

[0087] As can be seen from Table 7, 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 surgical biological patch and the preferred embodiment 12 of the digestive surgical biological patch are similar, but only the preferred embodiment 12 of the digestive surgical biological patch shows performance instability. Therefore, it is speculated that the performance instability of the biological patch may be due to other parameter factors besides the maximum tensile elongation that the inventors have not discovered, making it impossible to stably control the mechanical parameters of the biological patch, which is unsuitable for use as a surgical material.

[0088] The inventors further conducted mechanical tests on 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 tensile elongation of 10-15% at 80-140 mmHg. Unexpectedly, they 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 larger maximum tensile elongation but a smaller burst strength.

[0089] Test method for burst strength: Fix the edge of a vascular patch of appropriate area, and apply force at a speed of 100 mm / min to the vascular patch with a rigid spherical cap of Φ5 mm diameter facing the area corresponding to the spherical cap. Test the burst strength. See Table 9 for the experimental results.

[0090] Table 9: Bursting strength of 10 examples with tensile elongation of 10-15% at 80-140 mmHg

[0091] Table 9 shows that the bursting strength of the 10 embodiments with tensile elongation of 10-15% at 80-140 mmHg is 22-50 N.

[0092] In summary, it can be preliminarily concluded that controlling the maximum tensile elongation and elastic deformation rate helps to stably control the mechanical parameters of biological patches.

[0093] Based on the burst strength parameter, the relevant parameter ranges of the biological patches 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 suture bleeding and with stable mechanical properties are as follows: maximum tensile elongation 30.7-33.2%, single suture traction force 21.5-36.1N, elastic deformation rate 23.5-29.2%, elastic deformation rate as a percentage of maximum tensile elongation 77-88%, and burst strength 44-46N.

[0094] V. Optimization of Preparation Method

[0095] Based on the conclusions drawn from animal experiment 1, the preparation method was further optimized based on the preferred preparation methods of digestive surgery biological patch 15 and thoracic surgery biological patch 5 and 23.

[0096] 1. The 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.

[0097] Table 10: Main differences in the preparation methods of preferred embodiments 15 for digestive surgery biological patches and preferred embodiments 5 and 23 for thoracic surgery biological patches.

[0098] 2. Optimization of preparation method (Examples 1-12)

[0099] Step 1: Preprocessing

[0100] ①Immerse healthy bovine pericardial tissue slices in hypotonic Hank's solution, and repeatedly rinse and replace the hypotonic Hank's solution to fully swell and break down various cells in the tissue, thereby removing cell fragments, nuclei and organelles that have been swollen and broken down after decellularization.

[0101] ② Rinse the tissue slides treated above repeatedly with physiological saline for 70-150 minutes each time, changing the physiological saline each time. The total number of rinsing times depends on whether there are no morphological cells or cell components and cell debris under the microscope of the tissue slide. Perform quantitative determination of protein and nucleic acid until no soluble protein and nucleic acid can be detected.

[0102] Table 11: Rinse time with physiological saline

[0103] ③ Use a Tween 80 surfactant solution to remove phospholipids and non-structural proteins, as well as some tissue matrix molecules such as hyaluronic acid, various chondroitin sulfates, and mucopolysaccharides from the tissue slices.

[0104] ④ After soaking in a 0.05-0.1% glutaraldehyde solution for 3-3.5 hours for the first time, soak in a 1.4-3.5% glutaraldehyde solution for another 3-3.5 hours.

[0105] Table 12: Glutaraldehyde solution concentration and soaking time

[0106] Step 2, Chemical Modification

[0107] The pretreated tissue material was placed in Cr 3+ The ion concentration is 0.0625 mol / dm³.3 The solution of hydroxychromium with an OH / Cr ratio of 0.5 and a pH of 2-3 was subjected to the first water bath shaking under the following conditions: water bath shaking at 24-37℃ for 2-5 hours.

[0108] The pH value of the material treatment solution was tested, and the pH value was increased by 0.3-0.6 pH units with 10% NaHCO3 for a total of 2-4 times, with each increase being at least 0.1 pH. Then, a second water bath shaking was performed under the following conditions: shaking in a water bath at 36-45℃ for 2-5 hours to obtain a vascular biological patch in the form of a single layer with a thickness of about 1 mm.

[0109] Table 13: Water Bath Oscillation Conditions

[0110] 3. Mechanical Testing

[0111] (1) Detection method

[0112] Each embodiment yielded 10 biological patches.

[0113] Based on 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 traction force were determined.

[0114] A 4cm long and 1cm wide biological patch was clamped along its length onto a tensile testing machine. A tensile load was applied at a speed of 100mm / min to stretch the biological patch until it broke. A tensile curve was plotted, and the elastic deformation rate of the biological patch was calculated based on the elastic deformation segment of the tensile curve during the test.

[0115] Fix the edge of a vascular patch of appropriate size, and apply force at a speed of 100 mm / min to the vascular patch with a rigid spherical cap of Φ5 mm diameter facing the area corresponding to the spherical cap, and test the puncture strength.

[0116] (2) Parameter range

[0117] Table 14: Mechanical parameters of Examples 1-12

[0118] 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 rate of 15.1-29.4%, an elastic deformation rate accounting for 54-90% of the maximum tensile elongation, a single-line suture tensile force of 18.4-33.7N, and a bursting strength of 38-55N.

[0119] VI. Animal Experiments 2

[0120] 1. Experimental Design

[0121] Observe the bleeding at the suture site of the biological patch and the stability of the biological patch's mechanical parameters.

[0122] 2. Selection of laboratory animals

[0123] The experimental animals were 60 male New Zealand white rabbits, each weighing 2-3 kg, provided by Beijing Junwei Experimental Animal Breeding Center.

[0124] The experimental animals were kept in an acclimatization facility and quarantined for one week prior to surgery. Post-surgery, the animals were housed in the animal room. The facility temperature was maintained between 16-26℃, and each animal was kept in an individual cage. The cages were cleaned daily. Standardized feed was provided, and the animals had free access to food and water.

[0125] 3. Surgical plan

[0126] Refer to animal experiment 1.

[0127] 4. Experimental Results

[0128] (1) Among the experimental animals, only the experimental animals using the biological patch obtained in Example 6 showed bleeding at the suture site. The elastic deformation rate of the biological patch obtained in this example was relatively small as a percentage of the maximum tensile elongation (54%).

[0129] (2) The biological patch obtained in Example 10 showed tearing at the suture. The single-line suture tensile force (18.4 N) of the biological patch obtained in this example was relatively small. This is consistent with the experimental results of the pulsating flow experiment.

[0130] (3) Excluding experimental animals exhibiting tearing and bleeding, the biological patches in Examples 2, 4, and 12 showed instability in performance at weeks 4 and 8 post-surgery. This demonstrates that optimizing the preparation method significantly improved the stability of the mechanical parameters of the biological patches. A specific comparison of the mechanical parameters revealed that the biological patches obtained in Examples 2, 4, and 12 had higher maximum tensile elongation and / or lower burst strength. This further confirms that the range of values ​​for maximum tensile elongation and burst strength affects the stability of the mechanical parameters of the biological patches.

[0131] In summary, the biological patches obtained in Examples 1-12 have a low probability of causing bleeding at the suture site and performance instability. The relevant mechanical parameters of the biological patches obtained in Examples 1-12 are as follows: maximum tensile elongation 20.1-35.1%, elastic deformation rate 15.1-29.4%, elastic deformation rate as a percentage of maximum tensile elongation 54-90%, single-line suture tensile force 18.4-33.7N, and burst strength 38-55N.

[0132] Based on the parameter boundaries, further optimization is performed. 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 tensile force is 20.4-33.7N, and the bursting strength is 41-55N, the biological patch can effectively prevent bleeding at the suture site and address the problems of unstable performance and tearing at the suture site.

[0133] Based on Examples 1, 3, 5, 7-9, and 11, which demonstrate successful carotid endarterectomy, close fit between the biological patch and the anastomosis, and timely and effective control of bleeding at the anastomosis site and surrounding tissues, a further preferred biological patch is one 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-suture traction force of 20.8-33.7 N, and a burst strength of 41-54 N. This biological patch can more effectively prevent bleeding at the suture site and address the issues of unstable performance and tearing of the biological patch at the suture site.

[0134] VII. Clinical Trials

[0135] 1. Experimental Objective

[0136] The biological patches obtained in Examples 1, 3, 5, 7-9 and 11 were used to evaluate whether the "vascular biological patch" has the expected safety and efficacy in vascular repair.

[0137] 2. Test Content

[0138] The safety and efficacy of vascular biopatch for vascular repair were evaluated through a randomized, controlled, multicenter, non-inferiority clinical trial.

[0139] 3. Subject selection

[0140] (1) Selection criteria

[0141] ① Asymptomatic patients with preoperative imaging showing carotid artery stenosis ≥70% or symptomatic patients with carotid artery stenosis >50%, with CTA as the final diagnostic criterion before inclusion; ② Physical condition and vital signs meeting the surgical requirements; ③ Inclusion patients willing to comply with the trial protocol and capable of regular follow-up examinations; ④ Subjects understanding and / or their guardians voluntarily signing informed consent forms.

[0142] (2) Exclusion criteria

[0143] ① Patients with a life expectancy of less than 1 year; ② Patients who cannot tolerate anesthesia; ③ Patients who cannot undergo head and neck vascular CTA; ④ Patients who have experienced large-area stroke or myocardial infarction within the past 30 days; ⑤ Patients with coagulation disorders and contraindications to heparin and antiplatelet drugs; ⑥ Patients with a recent history of gastrointestinal bleeding and who are unable to undergo antiplatelet drug treatment; ⑦ Patients with large intracranial aneurysms that cannot be treated in advance or simultaneously; ⑧ Patients with chronic total occlusion without obvious symptoms of cerebral ischemia; ⑨ Patients with a history of intracranial hemorrhage (intracerebral, subarachnoid, subdural, or epidural) within the past 30 days; ⑩ Patients with severe liver, kidney, or circulatory system diseases. Severe dementia or mental disorders cannot be followed up in outpatient clinics; Those who have been in other clinical trials within the past 3 months or are currently participating in other clinical trials; The researchers believed that there were other reasons why the participants were not suitable for enrollment.

[0144] (3) Criteria and procedures for stopping trial / trial treatment

[0145] ① If a subject's condition continues to deteriorate during the study period, potentially leading to life-threatening events, and the doctor deems it necessary to stop the clinical study, then the clinical study for that case shall be terminated; ② If a subject develops certain comorbidities, complications, or special physiological or pathological changes during the study, making it unsuitable or impossible for them to continue the study; ③ If a subject is unwilling to continue the clinical study and requests termination of the clinical study from the attending physician, then the clinical study for that case may be terminated.

[0146] (4) The expected overall duration of the clinical trial and the rationale for its determination

[0147] This trial, from the first case to the last, will last 16 months. The rationale is that one year after vascular repair surgery, the efficacy and safety of the experimental product can be clearly observed. The enrollment period at each center is expected to be 4 months. During this process, the clinical trial is divided into two phases: first, a full summary is conducted 6 months after subject enrollment; second, clinical follow-up is completed 1 year or more after surgery.

[0148] (5) The expected duration of participation for each participant is 1 year.

[0149] (6) The number of subjects required for the clinical trial was 140 (10 subjects for each biological patch obtained in each embodiment), of which 70 were in the experimental group and 70 were in the control group. The control group used a vascular patch registered by AESCULA PAG of Aesculap Inc., made of polyurethane.

[0150] 4. Effectiveness Evaluation Methods

[0151] (1) Primary endpoint: For carotid artery stenosis, defined as >50% as indicated by postoperative carotid CTA at 1 year or more postoperatively. If no stenosis was observed in the subject, the treatment was considered effective, and the efficacy rate for each group was calculated. (2) Secondary endpoint: The incidence of perioperative complications after vascular biopatch angioplasty, including pseudoaneurysm rupture, new cranial nerve injury, postoperative myocardial infarction, postoperative heart failure, postoperative ischemic stroke, postoperative hemorrhagic stroke, incision hematoma, and TIA.

[0152] (Transient ischemic attack), postoperative wound. (3) Evaluation, recording and analysis of efficacy parameters were performed by CTA examination of the carotid arteries during visits 1, 3, 4 and 5. (4) Safety evaluation methods: ① Vital signs, laboratory indicators, neurological recovery status, and incidence of adverse events.

[0153] 5. Test Procedure

[0154] Table 15: Test Procedure Note: 1. Hemoglobin, white blood cell count, platelet count; 2. Total cholesterol, triglycerides, high-density lipoprotein, low-density lipoprotein, fasting blood glucose; 3. Prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), international normalized ratio of prothrombin time (PTINR).

[0155] 6. Clinical trial results

[0156] (1) 140 subjects were enrolled (70 in the experimental group and 70 in the control group), and 8 cases dropped out. Dropout refers to the number of subjects who did not complete the clinical trial.

[0157] (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 efficacy were clinically confirmed.

[0158] (3) No restenosis of the carotid artery occurred in the experimental group 1 year or more after surgery. Stenosis was defined as >50% as indicated by postoperative carotid CTA. It is believed that the use of biological patches for the treatment of subjects who underwent carotid endarterectomy is effective.

[0159] 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 pseudoaneurysm rupture, new cranial nerve injury, postoperative myocardial infarction, postoperative heart failure, postoperative ischemic stroke, postoperative hemorrhagic stroke, incision hematoma, and transient ischemic attack), neurological recovery, carotid vascular examination results, and brain tissue examination. This indicates that the treatment of subjects undergoing carotid endarterectomy using biological patches is safe.

[0160] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A vascular biological patch, characterized in that, The vascular bio-patch 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 38-55 N.

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

3. The vascular biological patch according to claim 1, characterized in that, The single-line suture traction force of the vascular biopatch is 20.4-33.7 N.

4. The vascular biological patch according to claim 1, characterized in that, The vascular bio-patch has 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 tensile force of 20.8-33.7N, and a bursting strength of 41-54N.

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

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

7. A method for preparing vascular biological patches, characterized in that, include: (1) After removing cellular components, phospholipids, non-structural proteins and immunogenic molecules, the bovine pericardium was soaked in 0.05-0.1% glutaraldehyde solution and 1.4-3.5% glutaraldehyde solution, respectively. (2) Placed in Cr 3+ The ion concentration is 0.0625 mol / dm³. 3 Immersion treatment with a hydroxychromium solution with an OH / Cr ratio of 0.5 and a pH of 2-3.

8. The method according to claim 7, characterized in that, In step (1), the bovine pericardium, after the cellular components, phospholipids, non-structural proteins and immunogenic molecules were removed, was soaked in a 0.05-0.1% glutaraldehyde solution for 3-3.5 hours, and then soaked in a 1.4-3.5% glutaraldehyde solution for 3-3.5 hours.

9. The method according to claim 7, characterized in that, In step (2), the Cr 3+ The ion concentration is 0.0625 mol / dm³. 3 After the first water bath shaking of a hydroxychromium solution with an OH / Cr ratio of 0.5 and a pH of 2-3, the pH is increased by 0.3-0.6, and then a second water bath shaking is performed.

10. The method according to claim 9, characterized in that, In step (2), the Cr 3+ The ion concentration is 0.0625 mol / dm³. 3 The temperature for the first water bath shaking in a hydroxychromium solution with an OH / Cr ratio of 0.5 and a pH of 2-3 is 24-37℃. The second water bath oscillation was carried out at 36-45℃.

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