Method for preparing a reinforced vascular graft with a sleeve of autologous residual limb-derived blood vessels and a vascular graft

Abstract

The application discloses a self-limb-source blood vessel graft reinforcing sleeve and a preparation method of the blood vessel graft, and belongs to the field of blood vessel surgical medical instruments. The reinforcing sleeve is a C-shaped open-loop tubular structure, and is sequentially provided with a proximal end anchoring area, an anastomosis covering area and a distal end anchoring area in the axial direction. The anastomosis covering area adopts a double-layer structure, and the inner layer is grafted with heparin and CD34 antibodies on the surface. Warm control self-locking buckle belts made of shape memory polymers are arranged on both sides of the longitudinal opening of the sleeve, and can be automatically closed and locked under the triggering of the body temperature of a human body. The method processes the discarded autologous blood vessels during amputation into blood vessel grafts on site, and then installs the sleeve by buckling from the side. The application does not need to be penetrated and sutured, is simple to operate, has the functions of anastomosis reinforcement, thrombosis resistance and endothelialization promotion, and the material can be gradually degraded, so that the problem that a traditional device is easy to damage blood vessels is effectively solved, and the application is suitable for amputation stump arteriovenous circulation reconstruction.

Description

Technical Field

[0001] This invention relates to the field of medical device technology, specifically to a method for preparing an autologous limb-derived vascular graft reinforcement cannula and vascular graft. Background Technology

[0002] According to statistics from the World Health Organization, there are over 40 million amputees worldwide, with more than 1.5 million new amputations performed annually, among which diabetes-related amputations account for the highest proportion. Post-amputation complications such as stump ischemic atrophy, bone resorption, delayed wound healing, and phantom limb pain occur in 50%-80% of cases, severely impacting patients' quality of life. Traditional amputation surgery often employs a "ligation and abandonment" approach to the blood vessels at the amputation plane, leading to three main problems: first, the stump tissue loses its direct blood supply, relying solely on collateral circulation for perfusion, exacerbating ischemia and atrophy; second, a large amount of natural autologous blood vessel resources in the abandoned limb segment are wasted; and third, the lack of an effective local blood circulation loop hinders tissue healing and remodeling.

[0003] In the field of vascular grafts, existing technologies mainly include autologous vein grafts and synthetic vascular grafts. Autologous great saphenous vein is the preferred material for vascular bypass, but it requires separate incision for harvesting, causing additional trauma and donor site complications. Synthetic vascular grafts (such as ePTFE) have a patency rate of only 40%-50% after 2 years in small-diameter applications, mainly because the material surface lacks natural endothelial coverage, making it prone to thrombosis, and its elastic modulus is much higher than that of natural arteries, leading to anastomotic compliance mismatch and intimal hyperplasia.

[0004] Regarding external support devices for vascular anastomosis, existing products such as the Israeli VasQ and FRAME have significant limitations: VasQ is used for arteriovenous fistulas in hemodialysis and is designed for high-flow maintenance (300-800 mL / min), lacking flow-limiting capabilities; FRAME is a circumferentially wrapped mesh sleeve, requiring the graft to be inserted into the mesh during surgery, which is cumbersome and necessitates suturing for fixation, increasing the risk of vascular injury. None of these products are designed to address the specific needs of low-flow circulation reconstruction in amputation stumps.

[0005] Shape memory polymers (SMPs) have been used in the medical device field, such as endovascular embolization devices and self-expanding stents. However, in external vascular support / self-locking devices, the technical solution of using body temperature to trigger the shape memory effect to achieve automatic closure and fixation of the cannula has not been reported before.

[0006] In summary, the existing technology has the following problems that urgently need to be solved: 1. Waste autologous blood vessel resources during amputation are not effectively utilized, and there is a lack of standardized methods for on-site processing into grafts; 2. Existing external vascular support devices are cumbersome to operate, requiring the insertion of a sheath and suturing for fixation, which can easily damage blood vessels; 3. There is a lack of integrated devices suitable for arteriovenous circulation reconstruction of amputation stumps, which also have the functions of anastomosis reinforcement, antithrombosis and automatic locking. Summary of the Invention

[0007] This invention provides a method for preparing a reinforcement cannula for autologous limb-derived vascular grafts and vascular transplants, which can solve the problems of ineffective utilization of discarded autologous vascular resources and the cumbersome operation and numerous limitations of existing vascular external support devices in the prior art.

[0008] To achieve the above objectives, in a first aspect, the present invention provides the following technical solution: an autologous limb-derived vascular graft reinforcement cannula, comprising a cannula body, the cannula body being generally tubular in structure, the cannula body having a longitudinal opening running the entire length along its axial direction, the opening angle being 30-60°, forming a C-shaped open-loop configuration, the cannula body having a proximal anchoring area, an anastomosis coverage area, and a distal anchoring area seamlessly connected sequentially along its axial direction, the cannula body including an outer layer, an inner layer disposed inside the outer layer of the anastomosis coverage area, the proximal anchoring area for wrapping the target artery, the distal anchoring area for wrapping the target vein, a vascular graft connecting the target artery and the target vein, the inner layer covering the anastomosis between the target artery, the target vein, and the vascular graft, and shape memory polymer temperature-controlled self-locking buckles fixed along the entire axial length of both sides of the longitudinal opening. The shape memory polymer temperature-controlled self-locking buckle has a glass transition temperature of 35-40℃. It is pre-programmed into an unfolded state at room temperature (20-25℃) to keep the longitudinal opening open. At body temperature (37℃), the shape memory effect is triggered, causing it to curl and close towards the midline of the cannula body, locking the cannula body into a near-closed loop. The C-shaped open-loop cannula body allows for lateral snap-fit ​​installation, eliminating the need to insert the vascular graft into the cannula, shortening the surgical time and avoiding damage to the anastomosis during insertion. The proximal and distal anchoring zones are respectively aligned with the arteries and veins, providing bidirectional anchoring. The anastomosis coverage area specifically covers the anastomosis, with clear functional zoning and strong targeting. The combination of the C-shaped open-loop cannula body and the shape memory polymer temperature-controlled self-locking buckle solves the pain points of cumbersome installation and large fixation trauma of traditional external support devices, achieving "one-click" automatic fixation.

[0009] Preferably, the outer layer is made of electrospun nanofibers blended with polycaprolactone and polyurethane, with a mass ratio of polycaprolactone to polyurethane of 60:40 to 70:30. PCL provides strength and controllable degradation, while PU provides elasticity. The elastic modulus after blending can be adjusted to 5-15 MPa, which is close to that of natural arteries, thus avoiding intimal hyperplasia caused by anastomotic compliance mismatch. The electrospun nanofibers form pores of 5-15 μm, allowing adventitia cells to grow in and achieving integrated healing between the cannula and the vessel wall.

[0010] Preferably, the inner layer is made of electrospun nanofibers blended with polycaprolactone and gelatin, with a mass ratio of polycaprolactone to gelatin of 50:50 to 70:30. Gelatin has a fast degradation rate, while PCL has a slow degradation rate. After blending, the inner layer is completely degraded in 3-6 months, which perfectly matches the endothelialization time window.

[0011] Preferably, the outer and inner layers are deposited layer by layer through electrospinning to form an integrated interface without adhesives. The molecules of the two layers permeate each other, resulting in high bonding strength, and the layers will not separate under vascular pulsation and tissue traction.

[0012] Preferably, the inner surface of the inner layer is uniformly distributed with heparin grafts and CD34 antibodies. Heparin grafts can continuously release heparin for more than 28 days, inhibiting platelet adhesion and aggregation, and reducing the risk of early thrombosis. CD34 antibodies can capture endothelial progenitor cells in circulating blood in situ, accelerate the endothelialization process of the anastomosis, and improve the long-term patency rate.

[0013] Preferably, the density of heparin grafting on the inner surface of the inner layer is 2-8 μg / cm³. 2 The cumulative release rate over 28 days was 60%-80%; the CD34 antibody density was 0.5-2.0 μg / cm³. 2 .

[0014] Preferably, the shape memory polymer temperature-controlled self-locking buckle has an L-shaped cross-section that bends towards the centerline of the sleeve body. When closed, the two buckles overlap to form a near-closed loop, resulting in a more secure fixation.

[0015] Secondly, the present invention also provides a method for preparing an autologous limb-derived vascular graft, comprising the following steps: S1. Vascular harvesting: During amputation, autologous vascular segments are separated from the discarded limb segment at the distal end of the amputation plane and immediately preserved in UW solution. S2. Vascular pretreatment: The lumen of the autologous vascular segment is flushed with a vascular irrigation device pre-filled with heparinized saline at a concentration of 100 U / mL. Excess loose connective tissue and adipose tissue on the outer surface of the vascular segment are removed under a surgical microscope, while the adventitia is preserved. S3. Preparation of roll-type variable diameter tube: The pre-treated autologous blood vessel segment is longitudinally cut along the long axis and flattened into a sheet patch. A sizing rod corresponding to the target inner diameter is selected. The sheet patch is rolled around the sizing rod with the inner membrane side facing the surface of the sizing rod to form a tube. The overlapping area of ​​the tube wall is intermittently or continuously sutured along the longitudinal edge with absorbable sutures. After the sizing rod is removed, the vascular graft is obtained. S4. Installation of reinforcement sleeve: Select the autologous limb vascular graft reinforcement sleeve as described in the first aspect, slide the sleeve along its longitudinal opening from the side of the anastomosis and snap it in, adjust the position of the sleeve so that the inner layer of the anastomosis coverage area completely covers the two anastomosis sites between the target artery, the target vein and the reel-type variable diameter tube. S5. Automatic locking of the cannula: During the waiting time, the shape memory polymer temperature-controlled self-locking buckle (3) triggers the shape memory effect at a body temperature of 37°C, curls and closes towards the midline of the cannula body, locks the cannula body into a near-closed ring, and completes the fixation of the reinforced cannula and the vascular graft.

[0016] It effectively utilizes discarded blood vessels from amputation to prepare grafts, eliminating the need for additional material harvesting trauma and avoiding donor site complications; the vascular graft is a roll-type variable diameter tube, solving the problem of arteriovenous diameter mismatch, and can prepare grafts of any inner diameter to suit the needs of different patients; the operation is simple, requires no sutures, significantly shortens the operation time, and reduces the difficulty of the operation.

[0017] Preferably, the UW solution described in step S1 contains 5 mmol / L adenosine, 1 mmol / L allopurinol, 3 mmol / L glutathione, 30 mmol / L raffinose, 100 mmol / L lactobionic acid, and 50 g / L hydroxyethyl starch, with a pH of 7.4 ± 0.05 and an osmotic pressure of 320 ± 10 mOsm / kg. The autologous vascular segment is preserved in the UW solution for no more than 4 hours. Adenosine can replenish ATP, allopurinol inhibits the generation of reactive oxygen species, glutathione has antioxidant properties, raffinose and lactobionic acid maintain osmotic pressure, and hydroxyethyl starch maintains colloidal osmotic pressure, thus comprehensively protecting the activity of vascular endothelial cells.

[0018] Preferably, the sizing bar is a stainless steel bar with a tetrafluoroethylene coating on its surface. The tetrafluoroethylene coating has a low coefficient of friction, which makes it easy to pull out the sizing bar after winding without damaging the vascular endothelium.

[0019] Compared with the prior art, the beneficial effects of the present invention are: 1. Transforming discarded blood vessels from amputated limbs into valuable resources without additional donor site trauma. This invention directly utilizes the autologous arteries and veins from the discarded limb segment during amputation surgery, processing them on-site into grafts. Unlike traditional autologous saphenous vein harvesting, which requires incisions at another site, this eliminates donor site trauma and the risk of complications. Autologous blood vessels have a natural endothelial cell lining, resulting in superior blood compatibility compared to any synthetic material.

[0020] 2. The C-shaped snap design eliminates the need for a sheath, significantly simplifying intraoperative procedures. The reinforcing cannula adopts a C-shaped open-loop structure, which can be directly snapped onto the periphery of the anastomosis from the side during the operation, without the need to insert the graft into the cannula. Compared with traditional full-circumference wrap-around external support devices, the installation operation is simpler and faster.

[0021] 3. The shape memory polymer temperature-controlled self-locking buckles on both sides of the C-shaped opening remain open at room temperature for easy installation and positioning. Upon contact with body temperature, they automatically trigger the shape memory effect to contract and close, with a closing time of 35-120 seconds and a radial adhesion force of 0.5-2.0N after closure. This self-locking mechanism eliminates the need for additional sutures or external fixation devices, reducing surgical steps and operation time.

[0022] 4. The inner layer of the anastomotic coverage area simultaneously possesses heparin covalent grafting and CD34 antibody modification, achieving dual functions of antithrombosis and endothelialization promotion. In vitro test results show that the platelet adhesion density of the dual-functional surface is reduced by approximately 90% compared to the unmodified control, and the EPC capture density reaches 152±22 cells / mm². 2 Within 14 days, the epidermal coverage rate reached 85.2 ± 5.5%.

[0023] 5. The inner layer of the anastomosis coverage area degrades completely within 3-6 months, matching the endothelialization time window; the outer layer degrades completely within 6-12 months, providing continuous mechanical support to the anastomosis until the vascular wall tissue remodeling is complete. The overall quality retention rate is <10% at 12 months, and no permanent foreign body residue is produced.

[0024] 6. After 4 hours of storage at 4°C, the modified UW solution provided in the kit maintained 82.3±3.2% cell viability, 2.8±0.4 points of endothelial integrity, and 7.5±2.0 μmol / L of NO release capacity in vascular tissue cells; while after 4 hours of storage in ordinary saline, cell viability was only 38.5±6.8%. Attached Figure Description

[0025] Figure 1 This is a front sectional view of the reinforced sleeve of the present invention; Figure 2 This is a side sectional view of the reinforced sleeve of the present invention; Figure 3 This is a cross-sectional view of the matching coverage area of ​​the present invention; Figure 4 This is a schematic diagram of the autologous blood vessel segment of the present invention; Figure 5 This is a schematic diagram of the sheet-like patch of the present invention; Figure 6 This is a schematic diagram illustrating the process of using the sizing bar of the present invention; Figure 7 This is a flowchart of the preparation method of the present invention.

[0026] Figure label: 1. Outer layer; 2. Inner layer; 3. Shape memory polymer temperature-controlled self-locking buckle; 4. Longitudinal opening; 5. Integrated interface; 6. Heparin graft; 7. CD34 antibody; 8. Patch; 10. Autologous vascular segment; 12. Diameter rod; 14. Absorbable suture; 100. Target artery; 200. Target vein; 300. Vascular graft; A. Proximal anchoring area; B. Anastomosis coverage area; C. Distal anchoring area. Detailed Implementation

[0027] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0028] like Figure 1-7 As shown, this invention addresses the problems of ineffective utilization of discarded autologous vascular resources and cumbersome operation and numerous limitations of existing vascular support devices in the prior art. The invention provides the following technical solution: an autologous limb-derived vascular graft reinforcement cannula, comprising a cannula body. The cannula body is generally tubular in structure, with a longitudinal opening 4 running the entire length along its axial direction. The opening angle is 30-60°, forming a C-shaped open-loop configuration. The cannula body is seamlessly connected axially to a proximal anchoring area A, an anastomosis coverage area B, and a distal anchoring area C. The cannula body includes an outer layer 1, and an inner layer 2 is disposed inside the outer layer 1 of the anastomosis coverage area B. The proximal anchoring area A is used to wrap the target artery 100, and the distal anchoring area C is used to wrap the target vein 200. A vascular graft 300 connects the target artery 100 and the target vein 200. The inner layer 2 covers the anastomosis between the target artery 100, the target vein 200, and the vascular graft 300. The longitudinal opening 4 has shape memory polymer temperature-controlled self-locking straps 3 fixed along its entire axial length on both sides of its two edges. The glass transition temperature of the shape memory polymer temperature-controlled self-locking straps 3 is 35-40℃. The shape memory polymer temperature-controlled self-locking straps 3 are pre-programmed into an unfolded state at room temperature (20-25℃) to keep the longitudinal opening 4 open. At body temperature (37℃), the shape memory effect is triggered, causing the straps to curl and close towards the centerline of the sleeve body, locking the sleeve body into a near-closed loop. The C-shaped open loop sleeve... The main body of the cannula can be installed with a lateral snap-fit, eliminating the need to insert the vascular graft into the cannula, thus shortening the operation time and avoiding damage to the anastomosis during insertion. The proximal and distal anchoring zones are respectively aligned with the arteries and veins, providing bidirectional anchoring. The anastomosis coverage area is specifically designed to cover the anastomosis, with clear functional zoning and strong targeting. The combination of the C-shaped open-loop cannula main body and the shape memory polymer temperature-controlled self-locking buckle 3 solves the pain points of traditional external support devices, such as cumbersome installation and large fixation trauma, and achieves "one-click" automatic fixation.

[0029] In this embodiment, the outer layer 1 is made of electrospun nanofibers blended with polycaprolactone (PCL) and polyurethane (PU). The mass ratio of PCL to PU is 60:40 to 70:30. PCL provides strength and controllable degradation, while PU provides elasticity. The elastic modulus after blending can be adjusted to 5-15 MPa, which is close to that of natural arteries, avoiding intimal hyperplasia caused by anastomotic compliance mismatch. The electrospun nanofibers form pores of 5-15 μm, allowing adventitia cells to grow in and achieving integrated healing between the cannula and the vessel wall. Both PCL and PU are FDA-approved medical materials, and their in vivo degradation products are non-toxic and do not cause inflammatory reactions. The electrospun nanofibers form pores of 5-15 μm, allowing adventitia cells to grow in and achieving integrated healing between the cannula and the vessel wall.

[0030] In this embodiment, the inner layer 2 is made of electrospun nanofibers blended with polycaprolactone and gelatin, with a mass ratio of polycaprolactone to gelatin of 50:50 to 70:30. Gelatin degrades rapidly, while PCL degrades slowly. The inner layer is completely degraded in 3-6 months after blending, perfectly matching the endothelialization time window. Gelatin contains RGD cell adhesion sites, promoting endothelial cell adhesion and proliferation. The surface of the PCL / gelatin nanofibers is rich in carboxyl and amino groups, facilitating the covalent grafting of heparin and CD34 antibodies.

[0031] In this embodiment, the outer layer 1 and the inner layer 2 are deposited layer by layer through electrospinning to form an integrated interface 5. There is no adhesive, and the molecules of the two layers permeate each other, resulting in high bonding strength. Under vascular pulsation and tissue traction, interlayer separation will not occur. There is no obvious boundary at the interface, which ensures the uniformity of the overall mechanical properties of the cannula.

[0032] In this embodiment, the inner surface of the inner layer 2 is uniformly distributed with heparin graft 6 and CD34 antibody 7. Heparin graft 6 can continuously release heparin for more than 28 days, inhibiting platelet adhesion and aggregation, and reducing the risk of early thrombosis. CD34 antibody 7 can capture endothelial progenitor cells in situ in circulating blood, accelerating the endothelialization process of the anastomosis and improving the long-term patency rate. The antithrombotic effect of heparin buys time for endothelialization, while the endothelialization-promoting effect of CD34 antibody fundamentally solves the thrombosis problem. The two work synergistically to achieve the goals of short-term antithrombosis and long-term patency. The density of heparin graft 6 on the inner surface of the inner layer 2 is 2-8 μg / cm³. 2 The cumulative release rate over 28 days is 60%-80%; the CD34 antibody 7 density is 0.5-2.0 μg / cm³. 2 If the density of heparin graft 6 is too low, the antithrombotic effect will be insufficient; if it is too high, it will easily lead to bleeding. If the density of CD34 antibody 7 is too low, the capture efficiency will be insufficient; if it is too high, it will increase costs and immunogenicity risks. (0.5-2.0 μg / cm³) 2 This is the range for optimal capture efficiency.

[0033] In this embodiment, the cross section of the shape memory polymer temperature-controlled self-locking buckle 3 is an L-shaped structure that bends towards the centerline of the sleeve body. When closed, the two buckles overlap each other to form a near-closed loop, making the fixation more secure.

[0034] The processing and fabrication process of the autologous limb-derived vascular graft reinforcement cannula in this embodiment includes the following steps: (1) Inner layer preparation: Prepare a PCL / gelatin mixed solution according to the ratio of each example, with a PCL:gelatin ratio of 50:50 to 70:30, using hexafluoroisopropanol (HFIP) as the solvent, a total concentration of 12% w / v, and magnetically stir for 12 hours until the solution is transparent and homogeneous. Electrospinning parameters: applied voltage 15-18kV, solution flow rate 0.5-1.0mL / h, receiving distance 15-20cm, and rotating collector speed 200-500rpm. After spinning, dry in a vacuum drying oven at 37°C for 24 hours to remove residual solvent.

[0035] (2) Outer layer preparation: Prepare a PCL / PU mixed solution according to the ratios in each example, with a PCL:PU ratio of 60:40 to 70:30, using HFIP as the solvent, and a total concentration of 15% w / v. Electrospinning parameters: applied voltage 18-22kV, solution flow rate 1.0-2.0mL / h, and braiding angle controlled at 45±5° by coordinating the collector rotation speed and lateral movement speed. The outer layer is directly deposited on the inner layer surface, forming an integrated bilayer structure.

[0036] (3) Heparin grafting: Heparin was covalently grafted onto the inner surface using the EDC / NHS coupling method. The nanofiber membrane was immersed in MES buffer (pH 5.5) containing 50 mM EDC and 25 mM NHS to activate the carboxyl groups for 2 hours, and then transferred to heparin sodium solution (1 mg / mL) for 4 hours at a reaction temperature of 4°C. After the reaction, the free heparin was thoroughly washed with deionized water to remove it.

[0037] (4) CD34 antibody modification: The heparin-grafted nanofiber membrane was immersed in a CD34 monoclonal antibody solution (concentration 10 μg / mL, dissolved in PBS buffer) and incubated at 37°C for 2 hours to allow the CD34 antibody to be fixed on the surface by electrostatic adsorption and covalent coupling through the heparin layer. After incubation, the membrane was washed three times with PBS buffer to remove unbound antibodies.

[0038] (5) Preparation of SMP ring and buckle bands: SMP polyurethane material with adjustable Tg was selected, and ring and buckle band blanks were prepared by hot pressing. The blanks were heated to Tg+20°C, and a permanent shape (closed state) was formed in the mold. After cooling to room temperature, they were removed. Then, they were heated again to Tg+10°C, stretched or bent to a temporary shape (unfolded state), and rapidly cooled to below Tg to fix the temporary shape.

[0039] (6) Assembly: The proximal anchoring region, the anastomosis coverage region (double-layer nanofiber membrane), and the distal anchoring region are arranged sequentially on the rotating mandrel. The proximal and distal anchoring regions can be spun separately and then joined together, while the anastomosis coverage region is prepared by layer-by-layer deposition. The SMP buckle is fixed with medical adhesive on both sides of the longitudinal opening along the axial direction. After assembly, a C-shaped longitudinal opening is cut along the predetermined position.

[0040] As a supplement to this embodiment, a flow-limiting ring area is added between the proximal anchoring zone A and the anastomosis coverage zone B of the basic type casing, forming a four-segment structure of "proximal anchoring zone A → flow-limiting ring area → anastomosis coverage zone B → distal anchoring zone C". The flow-limiting ring area is nested with an SMP adjustable ring band, and the rest of the structure is completely consistent with the basic type.

[0041] The SMP adjustable ring band has a three-layer composite structure, from the outside to the inside: Outer SMPU shape memory skeleton: It is injection molded using DiAPLEXMM3520 (Tg=35℃). There are 28 unidirectional ratchet teeth evenly distributed around the outer edge, with a tooth depth of 0.4mm. Each ratchet tooth corresponds to a 0.5mm narrowing of the tube diameter, which is adjustable in 5 levels, namely an open inner diameter of 4.0mm and a fully narrowed inner diameter of 2.0mm.

[0042] Middle layer PEEK pawls: Three medical-grade PEEK (InvibioPEEK-OPTIMA) pawls are evenly distributed at 120°, which work with the ratchet to form a one-way locking mechanism that can only tighten and cannot loosen.

[0043] Inner ePTFE smooth lining: using Gore-Tex microporous membrane, 30μm thick, with a node spacing of 0.6μm, attached to the inner surface of the ring to prevent thrombosis and damage to the vessel wall.

[0044] When in use, after the reinforcing sleeve is locked, connect the ultrasonic flow meter to monitor the arterial and venous circulation flow in real time.

[0045] Take a dedicated gear wrench, align the U-shaped groove of the wrench head with the exposed gear mark, and move it circumferentially gear by gear. Each gear movement narrows the pipe diameter by 0.5mm, and the flow rate decreases stepwise according to the Hagen-Poiseuille equation.

[0046] Adjust the flow rate to a stable range of 80-120 mL / min, wait 90 seconds, and the SMPU skeleton will complete its shape recovery at body temperature, precisely locking the tube diameter within ±0.1 mm accuracy.

[0047] The method for preparing the vascular graft in this embodiment includes the following steps: Step 1: Blood vessel sampling.

[0048] During amputation, autologous vascular segments 10 were obtained from the discarded limb segment distal to the amputation plane. The autologous vascular segments were selected from arteries and / or veins in the discarded limb segment, with a harvested length of 30-150 mm (40 mm in Example 1, 60 mm in Example 2, 90 mm in Example 3, and 130 mm in Example 4), and an outer diameter of 2-8 mm (2.5 mm in Example 1, 3.5 mm in Example 2, 5 mm in Example 3, and 7 mm in Example 4). During dissection, sharp dissection was performed along the loose connective tissue layer between the adventitia and surrounding tissue, preserving the integrity of the adventitia. Immediately after dissection, the vascular segments were immersed in UW solution at a temperature of 4±1°C for preservation. The modified UW solution contains adenosine 5 mmol / L, allopurinol 1 mmol / L, glutathione 3 mmol / L, raffinose 30 mmol / L, lactobionic acid 100 mmol / L, and hydroxyethyl starch 50 g / L. The pH is 7.4 ± 0.05, and the osmotic pressure is 320 ± 10 mOsm / kg. It should be stored for no more than 4 hours.

[0049] Step two, vascular irrigation.

[0050] Using a vascular irrigation device pre-filled with heparinized saline at a concentration of 100 U / mL, insert it into one end of the autologous vessel segment 10 and irrigate the lumen at an irrigation pressure of 80-120 mmHg (80 mmHg in Example 1, 90 mmHg in Example 2, 100 mmHg in Example 3, and 120 mmHg in Example 4). The irrigation time should be no less than 30 seconds (30 seconds in Example 1, 45 seconds in Example 2, 60 seconds in Example 3, and 90 seconds in Example 4). The temperature of the irrigation fluid should be 4-8°C (4°C in Example 1, 5°C in Example 2, 6°C in Example 3, and 8°C in Example 4). Irrigate until the outflow is clear and free of blood clots and residue. During irrigation, check for any collateral ruptures; if any are found, repair them by ligation with 7-0 to 9-0 absorbable sutures.

[0051] Step 3, outer membrane trimming.

[0052] Under a surgical microscope or magnifying glass, excess loose connective tissue and adipose tissue on the outer surface of the blood vessel are removed with microsurgical scissors, preserving the adventitia itself. The thickness of the vessel wall after trimming is 0.3-0.8 mm (0.35 mm in Example 1, 0.45 mm in Example 2, 0.55 mm in Example 3, and 0.70 mm in Example 4). During the trimming process, the vessel surface is continuously moistened with heparinized saline at 4-8°C to prevent tissue drying and intimal damage.

[0053] Step 4: Graft morphology processing.

[0054] Choose one or more of the following processing methods based on the needs during the operation: Method A – Preparation of tubular graft 300: Cut the blood vessel segment processed in steps two and three to the required length, with a cutting length of 15-80 mm (20 mm in Example 1, 35 mm in Example 2, 50 mm in Example 3, and 70 mm in Example 4). Confirm that the intima side faces the lumen side, and the inner diameter of the tubular graft is 2-6 mm (2 mm in Example 1, 3 mm in Example 2, 4 mm in Example 3, and 6 mm in Example 4).

[0055] Method B – Preparation of Sheet Patch 8: Insert a longitudinally slicing guide into the lumen from one end of the blood vessel. Using microsurgical scissors, longitudinally slice the entire blood vessel wall along the long axis of the vessel, guided by the guide. After slicing, lay the blood vessel sheet flat on a moistened gauze with the inner membrane facing upwards. Trim the sheet into rectangular or elliptical patches with a length of 10-40 mm (12 mm in Example 1, 20 mm in Example 2, 30 mm in Example 3, and 38 mm in Example 4), a width of 3-12 mm (4 mm in Example 1, 6 mm in Example 2, 8 mm in Example 3, and 11 mm in Example 4), and a thickness of 0.3-0.8 mm.

[0056] Method C – Preparation of Roll-Rolled Reducing Tube: Take the sheet patch 8 prepared in Method B, select a sizing rod 12 corresponding to the target inner diameter, and roll the sheet patch 8 around the sizing rod with the inner membrane facing towards the surface of the sizing rod to form a tube. The width of the overlapping area of ​​the tube wall is 1-3mm (1.0mm in Example 1, 1.5mm in Example 2, 2.0mm in Example 3, and 2.5mm in Example 4). Use 7-0 to 9-0 absorbable sutures 14 to perform intermittent or continuous suturing along the longitudinal edge, with a suture spacing of 1-2mm (1.0mm in Example 1, 1.2mm in Example 2, 1.5mm in Example 3, and 2.0mm in Example 4). After suturing, remove the sizing rod to obtain a roll-rolled reducing tube with a finished inner diameter of 2-6mm and a wall thickness of 0.6-1.6mm.

[0057] Step 5: Graft quality testing.

[0058] The prepared grafts were subjected to a flushing patency test by injecting heparinized saline at a pressure of 80-120 mmHg to confirm that the lumen was patent and leak-free. The leakage rate at the suture of the roll-type reducing tube was <5% (2.1% in Example 1, 2.8% in Example 2, 3.5% in Example 3, and 4.2% in Example 4). The flatness and thickness uniformity of the patch 8 were visually inspected. After passing the tests, the grafts were immersed in modified UW solution at 4±1°C for no more than 2 hours (0.5 hours in Example 1, 1 hour in Example 2, 1.5 hours in Example 3, and 2 hours in Example 4).

[0059] The performance of the vascular grafts produced in the above embodiments is tested below: (a) Mechanical property testing: Tensile test: Using an Instron 5943 high-performance mechanical testing machine, the specimens were cut into rectangular strips of 40mm × 5mm, fixed on a fixture with a clamping distance of 20mm, and subjected to uniaxial tension at a tensile rate of 10mm / min until fracture. The stress-strain curves were recorded, and the ultimate tensile strength and elastic modulus were calculated. Five parallel samples were tested in each group (n=5).

[0060] Bursting pressure test: Fix both ends of the tubular sample to the sealing joint, connect one end to the injection pump, and continuously pressurize the cavity at a rate of 5 mmHg / s. Seal the other end and record the pressure in the cavity in real time through the pressure sensor until the sample ruptures. The peak pressure at the time of rupture is the bursting pressure.

[0061] Suture retention force test: 7-0 absorbable suture 14 is passed through the entire thickness of the specimen 2 mm from the edge of the specimen. One end of the suture is fixed to the upper clamp of the mechanical testing machine, and the other end of the specimen is fixed to the lower clamp. The suture is pulled upward at a tensile rate of 120 mm / min, and the peak force when the suture is pulled out of the specimen is recorded.

[0062] Radial compression test: The tubular specimen is placed between parallel plates and radially compressed at a compression rate of 1 mm / min. The stress value when the specimen is compressed by 50% is recorded as the radial compression strength.

[0063] Table 1 Mechanical properties of tubular grafts Table 2 Mechanical properties of sheet patches Table 3 Performance of Reducing Tubes (II) Mechanical performance testing of reinforced casing: Radial compressive strength, longitudinal tensile strength, and suture retention force were tested on the reinforced sleeve anastomosis coverage area, using the same testing methods as above.

[0064] Table 4 Mechanical properties of the reinforced casing splice coverage area (III) Antithrombotic and endothelialization performance tests: Platelet adhesion test: Fresh human platelet-rich plasma was collected, and each sample (1cm×1cm) was immersed in PRP and incubated at 37°C for 2 hours. After incubation, the samples were removed and gently rinsed three times with PBS buffer to remove unadhered platelets. The samples were fixed with 2.5% glutaraldehyde, dehydrated with graded ethanol, critically dried, and then sputter-coated with gold. The number of adhered platelets was counted by randomly selecting five fields of view under 2000x magnification using a scanning electron microscope (Hitachi SU8010).

[0065] EPC capture test: Preparation of human umbilical vein blood-derived EPC suspension (concentration 1×10⁻⁶) 5 (cells / mL), each group of tubular samples was connected to a dynamic perfusion circuit, and subjected to a shear force of 5 dyn / cm at 37°C. 2 Dynamic perfusion was performed for 2 hours. After perfusion, unbound cells were removed by PBS rinsing, and adherent cells were labeled with DiI fluorescence. The EPC capture density in each field of view was counted under a fluorescence microscope.

[0066] Endothelial coverage assay: EPCs were seeded onto the surface of each group of samples and statically cultured for 14 days before CD31 immunofluorescence staining. The primary antibody was mouse anti-human CD31 monoclonal antibody, and the secondary antibody was Alexa Fluor 488-labeled goat anti-mouse IgG. Cell nuclei were counterstained with DAPI. Imaging was performed using a confocal laser scanning microscope (Zeiss LSM880), and the percentage of endothelial coverage area was analyzed using ImageJ software.

[0067] Table 5. Antithrombotic and endothelialization properties (iv) In vitro degradation performance test: The inner and outer layer samples were cut into 10mm × 10mm square pieces. The initial dry weight was measured and the samples were placed in sealed centrifuge tubes containing 10mL of PBS buffer (pH 7.4). The tubes were then placed in a 37°C constant-temperature shaker (60rpm), with the PBS buffer replaced weekly. Three parallel samples were taken at each of the 1, 3, 6, 9, and 12-month time points. After rinsing with deionized water, the samples were vacuum-dried at 37°C to constant weight, and the mass retention rate was calculated. Simultaneously, tensile tests were performed on the residual samples at each time point, and the mechanical strength retention rate (residual tensile strength / initial tensile strength × 100%) was calculated.

[0068] Table 6. In vitro degradation performance (PBS at 37°C, pH 7.4) (v) SMP temperature control self-locking performance test: Glass transition temperature (Tg) determination: Using a differential scanning calorimeter (DSC, TA Instruments Q2000), 5-10 mg of SMP sample was sealed in an aluminum crucible and heated from 0°C to 80°C at a heating rate of 10°C / min. The heat flow curve was recorded, and Tg was determined by the midpoint temperature of the endothermic step.

[0069] Shape recovery rate test: The SMP buckle was programmed into a temporary shape (unfolded state, initial angle θ0 was recorded), and then the buckle was placed in a water bath at 37°C (near or above Tg) to observe its spontaneous recovery and closure process. After recovery was completed, the final angle θ was measured. f Shape recovery rate = (θ0 - θ) f ) / θ0×100%.

[0070] Locking force test: The reinforcing sleeve is fitted onto a silicone tube (simulating the surface of a blood vessel) with a matching outer diameter. After the SMP buckle is fully closed in a 37°C water bath, the sleeve is pulled out along the axial direction at a rate of 1 mm / min using a tension gauge. The peak force when the sleeve is removed from the silicone tube is recorded as the locking force (radial adhesion force).

[0071] Table 7 SMP Temperature Control Self-Locking Performance (vi) Preservation effect test of vascular preservation solution: Cell viability assay: Cell viability of vascular tissue was detected using the CCK-8 assay. Vascular fragments were harvested at each time point and incubated in serum-free medium containing 10% CCK-8 reagent at 37°C for 2 hours. The supernatant was then measured at 450 nm. The percentage of cell viability at each time point was calculated with 0 hour as 100% baseline.

[0072] Endothelial integrity scoring: Blood vessel segments were taken at each time point, stained with hematoxylin and eosin (HE), and the integrity of the endothelial layer was observed under an optical microscope. The score was based on a scale of 0 to 4 (4 points = continuous and complete endothelial cell coverage, 3 points = local endothelial loss <25%, 2 points = endothelial loss 25%-50%, 1 point = endothelial loss 50%-75%, 0 points = complete endothelial detachment).

[0073] NO release capacity assay: Vascular fragments were harvested at various time points and placed in Krebs buffer containing L-arginine. After incubation at 37°C for 1 hour, the supernatant was collected. The concentration of nitrite in the supernatant was detected using the Griess method, indirectly reflecting the NO release function of endothelial cells.

[0074] Table 8 Preservation Effect of Vascular Preservation Solution It should be noted that all directional indicators (such as up, down, left, right, front, back, etc.) in the embodiments of the present invention are only used to explain the relative positional relationship and movement of each component in a certain specific posture (as shown in the figure). If the specific posture changes, the directional indicator will also change accordingly.

[0075] Furthermore, in this invention, descriptions involving "first," "second," etc., are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly and specifically defined.

[0076] In this invention, unless otherwise explicitly specified and limited, the terms "connection," "fixed," etc., should be interpreted broadly. For example, "fixed" can mean a fixed connection, a detachable connection, or an integral part; it can mean a mechanical connection or an electrical connection; it can mean a direct connection or an indirect connection through an intermediate medium; it can mean the internal communication of two components or the interaction between two components, unless otherwise explicitly limited. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0077] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

Claims

1. A reinforcing cannula for autologous limb-derived vascular grafts, characterized in that, The device includes a cannula body, which is tubular in shape. The cannula body has a longitudinal opening (4) running the entire length along its axial direction, forming a C-shaped open-loop configuration. The cannula body is seamlessly connected axially to a proximal anchoring area (A), an anastomosis coverage area (B), and a distal anchoring area (C). The cannula body includes an outer layer (1), and an inner layer (2) is provided inside the outer layer (1) of the anastomosis coverage area (B). The proximal anchoring area (A) is used to wrap the target artery (100), and the distal anchoring area (C) is used to wrap the target vein (200). A vascular graft connects the target artery (100) and the target vein (200). The plant (300), the inner layer (2) covers the anastomosis between the target artery (100), the target vein (200) and the vascular graft (300), the two sides of the longitudinal opening (4) are fixed with shape memory polymer temperature-controlled self-locking buckles (3) along the entire axial length, the glass transition temperature of the shape memory polymer temperature-controlled self-locking buckles (3) is 35-40℃, the shape memory polymer temperature-controlled self-locking buckles (3) are pre-programmed into an unfolded state at room temperature 20-25℃ to keep the longitudinal opening (4) open; at body temperature 37℃, the shape memory effect is triggered, and the buckles curl and close towards the midline of the cannula body, locking the cannula body into a near-closed loop.

2. The autologous limb-derived vascular graft reinforcement cannula according to claim 1, characterized in that: The outer layer (1) is made of electrospun nanofibers blended with polycaprolactone and polyurethane, with a mass ratio of polycaprolactone to polyurethane of 60:40 to 70:

30.

3. The autologous limb-derived vascular graft reinforcement cannula according to claim 2, characterized in that: The inner layer (2) is made of electrospun nanofibers of polycaprolactone and gelatin, with a mass ratio of polycaprolactone to gelatin of 50:50 to 70:

30.

4. The autologous limb-derived vascular graft reinforcement cannula according to claim 3, characterized in that: The outer layer (1) and the inner layer (2) are deposited layer by layer through electrospinning to form an integrated interface (5).

5. The autologous limb-derived vascular graft reinforcement cannula according to claim 4, characterized in that: The inner surface of the inner layer (2) is uniformly covered with heparin grafts (6) and CD34 antibodies (7).

6. The autologous limb-derived vascular graft reinforcement cannula according to claim 5, characterized in that: The density of heparin grafts (6) on the inner surface of the inner layer (2) is 2-8 μg / cm³. 2 The cumulative release rate over 28 days was 60%-80%; the CD34 antibody (7) density was 0.5-2.0 μg / cm³. 2 .

7. The autologous limb-derived vascular graft reinforcement cannula according to claim 1, characterized in that: The cross section of the shape memory polymer temperature-controlled self-locking buckle (3) is an L-shaped structure that bends toward the centerline of the sleeve body.

8. A method for preparing an autologous limb-derived vascular graft, characterized in that: Includes the following steps: S1. Vascular sampling: During amputation, autologous vascular segments (10) are separated from the discarded limb segments at the distal end of the amputation plane of the residual limb. Immediately after separation, the autologous vascular segments (10) are immersed in UW solution for preservation. S2. Pretreatment of blood vessels: The lumen of the autologous blood vessel segment (10) was flushed with a blood vessel flushing and perfusion device pre-filled with heparinized saline at a concentration of 100 U / mL. Excess loose connective tissue and adipose tissue on the outer surface of the blood vessel were removed under a surgical microscope, while the adventitia was preserved. S3. Preparation of roll-type variable diameter tube: The pretreated autologous blood vessel segment (10) is longitudinally cut along the long axis and flattened into a sheet patch (8). A sizing rod (12) corresponding to the target inner diameter is selected. The sheet patch (8) is rolled around the sizing rod with the inner membrane facing the surface of the sizing rod to form a tube. The overlapping area of ​​the tube wall is intermittently or continuously sutured along the longitudinal edge with absorbable sutures (14). After the sizing rod (12) is removed, a vascular graft (300) is obtained. S4. Installation of reinforcement sleeve: Select the autologous limb vascular graft reinforcement sleeve as described in any one of claims 1-7, and insert the sleeve from the side of the anastomosis along its longitudinal opening (4). Adjust the position of the sleeve so that the inner layer (2) of the anastomosis coverage area (B) completely covers the two anastomosis sites between the target artery (100), the target vein (200) and the reel-type variable diameter tube. S5. Automatic locking of the cannula: During the waiting time, the shape memory polymer temperature-controlled self-locking buckle (3) triggers the shape memory effect at a body temperature of 37°C, curls and closes towards the midline of the cannula body, locks the cannula body into a near-closed ring, and completes the fixation of the reinforced cannula and the vascular graft (300).

9. The method for preparing autologous limb-derived vascular grafts according to claim 8, characterized in that: The UW solution described in step S1 contains 5 mmol / L adenosine, 1 mmol / L allopurinol, 3 mmol / L glutathione, 30 mmol / L raffinose, 100 mmol / L lactobionic acid and 50 g / L hydroxyethyl starch, with a pH of 7.4 ± 0.05 and an osmotic pressure of 320 ± 10 mOsm / kg. The autologous blood vessel segment (10) is stored in the UW solution for no more than 4 hours.

10. The method for preparing autologous limb-derived vascular grafts according to claim 8, characterized in that: The sizing bar (12) is a stainless steel bar with a tetrafluoroethylene coating on its surface.

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