A method for preparing a growing living homograft valve for replacement of a child's valve and its use

Abstract

The application provides a preparation method of a growing living homograft valve for child valve replacement and application thereof, and belongs to the field of biomedical technology. The application provides the preparation method of the growing living homograft valve for child valve replacement based on the clinical urgent demand of child valve replacement, takes the homograft donor heart as a source, and realizes standard donor screening, rapid off-body acquisition, complete valve root structure cutting, low-temperature activity protection and individualized modification forming, so that the valve endothelial cells, interstitial cells and natural extracellular matrix structure are retained to the maximum extent, the growing living homograft valve has sustained function and potential growth ability after implantation, the clinical problem that the existing child valve replacement technology cannot expand synchronously with growth and needs repeated operation replacement is solved, a growing valve replacement scheme with long-term application prospect is provided for children, especially newborns and infants, so that the reoperation rate is reduced, the long-term prognosis is improved and the life quality is improved.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, specifically relating to a method for preparing a growable living allogeneic valve for pediatric valve replacement and its application. Background Technology

[0002] Congenital valvular heart disease in children, especially severe aortic stenosis, pulmonary atresia, common truncus arteriosus, and other complex congenital outflow tract malformations, is a major cause of severe heart failure and death in newborns and infants. For these patients, surgical valve replacement is currently the primary treatment. Commonly used valve replacement options include mechanical valves, bioprosthetic valves, and cryopreserved allogeneic valves. While mechanical valves offer good long-term durability, their biggest drawback is their inability to grow synchronously with the child's development, requiring long-term anticoagulation therapy post-surgery, significantly increasing the risk of thrombosis and bleeding. Bioprosthetic valves, although avoiding long-term anticoagulation, are prone to early structural degeneration and calcification in pediatric patients, resulting in poor long-term durability. Cryopreserved allogeneic valves are currently a commonly used option for right ventricular outflow tract reconstruction in children, especially infants. However, due to the decellularization and cryopreservation process, the graft lacks viable cell components and cannot expand synchronously with the child's growth, often requiring multiple replacement surgeries during the child's growth, severely impacting long-term prognosis. Summary of the Invention

[0003] The purpose of this invention is to provide a method for preparing a growing living allogeneic valve for pediatric valve replacement. Using an allogeneic donor heart as the source, the method involves standardized donor screening, rapid ex vivo acquisition, complete valve root structure resection, low-temperature viability preservation, and individualized shaping. This method maximizes the preservation of valve endothelial cells, interstitial cells, and the natural extracellular matrix structure, enabling the graft to have continuous function and potential growth capacity after implantation. This solves the key clinical problem in existing pediatric valve replacement technologies that "cannot expand synchronously with growth and require repeated surgical replacements."

[0004] This invention provides a method for preparing a living, allogeneic valve, comprising the following steps: After rinsing the isolated donor heart with pre-cooled preservation solution, the target intact valve tissue was excised under aseptic conditions and then placed in a sterile environment at 0~8℃ for low-temperature viability protection. Before the implantation surgery, the target intact valve tissue is individually trimmed and shaped according to the child recipient's age, weight, valve annulus diameter and outflow tract anatomy, to obtain a living allogeneic valve that can grow.

[0005] Preferably, the target valve tissue includes at least one of the following structures: aortic root, pulmonary artery root, and double valve root structure.

[0006] Preferably, the scope of the target valve tissue to be harvested includes the complete valve leaflet, complete valve annulus, at least one complete valve sinus, continuous proximal ventricular myocardial border or annulus fibrosus tissue, and continuous distal ascending aortic wall or aortopulmonary artery wall tissue.

[0007] Preferably, the proximal tissue retention length of the target valve tissue is 1~5mm; The distal vessel wall of the target valve tissue is preserved for a length of 3-20 mm.

[0008] Preferably, the donor heart is sourced from brain-dead donor hearts, circulatory-dead donor hearts, domino donor hearts, and donor hearts that are not suitable for whole-heart transplantation but have intact valve structure and normal function.

[0009] Preferably, the donor heart also includes functional testing; The functional tests include echocardiography and / or in vitro visual examination to confirm the integrity of the semilunar valve structure, the good mobility of the leaflets, and the presence of obvious calcification, tearing, infectious vegetations, or congenital developmental abnormalities.

[0010] Preferably, the time for the donor heart to leave the body is controlled within 30 minutes.

[0011] Preferably, the preservation solution includes any one of the following: University of Wisconsin preservation solution, HTK solution, and Celsior solution.

[0012] Preferably, the growable biocompatible valve further includes pre-implantation quality assessment; The preimplantation quality assessment method includes at least one of the following: appearance integrity inspection, leaflet mobility assessment, occlusion integrity test, and sterility test.

[0013] The present invention also provides the application of the growable living allogeneic valve prepared by the above preparation method in the preparation of biomaterials for treating congenital heart valve diseases in children.

[0014] This invention provides a method for preparing a growable living allogeneic valve, including donor heart acquisition, target valve tissue harvesting, cryogenic viability preservation, and individualized shaping. The resulting growable living allogeneic valve can be used for surgical replacement of aortic and pulmonary valves in children. The preparation method of this invention gives the growable living allogeneic valve the following characteristics: 1. It possesses continuous growth potential, significantly reducing the need for multiple reoperations during childhood. Compared to existing mechanical valves, bioprosthetic valves, and cryopreserved allogeneic valves, which cannot grow synchronously with the child's physical development, this invention obtains and prepares allogeneic living valve tissue that retains cell viability. This allows the graft to retain the activity of valvular interstitial cells and surface endothelial cells after implantation, thus possessing the potential to expand the valve annulus and elongate the leaflets as the recipient grows. This preparation method is expected to significantly reduce the risk of repeated surgical replacements for pediatric patients due to valve size mismatch during growth and development. 2. It significantly improves the long-term durability of the graft and reduces the risk of structural degradation. Traditional cryopreserved or decellularized allogeneic valves are prone to leaflet fibrosis, calcification, and structural degeneration postoperatively due to the lack of viable cells, especially in infants and young children where the degeneration is faster. This invention, however, employs a fresh acquisition and low-temperature viability preservation process, avoiding decellularization, chemical fixation, and cryo-deactivation steps. This maximizes the preservation of the valve's natural tissue structure and extracellular matrix integrity, thereby improving the long-term mechanical stability and tissue durability of the graft and reducing the risk of early failure. 3. Closer to natural physiological structure, significantly improving postoperative hemodynamics in children. Compared to traditional artificial valves, which cannot fully mimic the geometry and hemodynamic characteristics of the natural semilunar valve, the living allogeneic valve prepared by this invention completely preserves the leaflets, annulus, sinus, and adjacent vessel wall structures. This allows for better reconstruction of the natural outflow tract anatomy, reducing the risk of postoperative transvalvular pressure gradient increase, valvular stenosis, or regurgitation, thus improving postoperative cardiac function recovery. 4. Expanding donor sources and increasing the utilization rate of donor heart tissue. Compared to traditional whole-heart transplantation, which has high requirements for donor heart function and limited donor sources, this invention can utilize donor hearts that are not originally suitable for whole-heart transplantation but have intact valve structure and function. It is particularly suitable for brain-dead donors, circulatory-dead donors, and domino donors, thereby significantly improving donor tissue utilization and alleviating the shortage of pediatric valve replacement donors. 5. Supports individualized and precise matching for children, improving clinical fit. Addressing the significant differences in age, weight, and valve annulus diameter among children, this invention can individually trim and size the donor valve root according to the recipient's anatomy, meeting the replacement needs of newborns, infants, and children at different stages, improving graft implantation success rate and long-term fit. 6. Helps reduce the risk of long-term anticoagulation and related complications.Compared to mechanical valves, which require long-term anticoagulation and carry risks of thrombosis and bleeding, the living allogeneic valve prepared in this invention is a natural biological tissue with better biocompatibility and endothelial coverage potential. It can avoid long-term systemic anticoagulation therapy in clinical practice, reducing the long-term medication burden and related complications in pediatric patients. Therefore, this invention provides a valve replacement solution that possesses good biocompatibility and can continuously grow and maintain stable function in children over a long period. This invention uses freshly obtained and prepared allogeneic living valve grafts with preserved cell viability, showing a clear growth trend in three clinical cases. The average valve diameter immediately after surgery was 9.2 mm, increasing to 15.7 mm at 26 months, an increase of approximately 70.7%, indicating a continuous growth capacity not found in traditional techniques. Therefore, compared to existing pediatric valve replacement techniques, this invention has significant advantages in reducing the need for repeat surgeries, improving long-term fit, and improving long-term prognosis in children. This invention provides a growth-type valve replacement solution with long-term application prospects for children, especially newborns and infants, thereby reducing the reoperation rate, improving long-term prognosis and improving quality of life, and has clear clinical translational value and application prospects. Attached Figure Description

[0015] Figure 1 To follow up on the changes in the diameter of the transplanted valve in 3 patients; Figure 2 This is an incomplete valve tissue sample. Detailed Implementation

[0016] This invention provides a method for preparing a living, allogeneic valve, comprising the following steps: After rinsing the isolated donor heart with pre-cooled preservation solution, the target intact valve tissue was excised under aseptic conditions and then placed in a sterile environment at 0~8℃ for low-temperature viability protection. Before the implantation surgery, the target intact valve tissue is individually trimmed and shaped according to the child recipient's age, weight, valve annulus diameter and outflow tract anatomy, to obtain a living allogeneic valve that can grow.

[0017] In this invention, the acquisition of the donor heart preferably includes functional testing. This functional testing preferably includes echocardiography and gross examination to confirm that the donor aortic or pulmonary valve is structurally intact, with good leaflet opening and closing function, and without significant calcification, leaflet tears, infectious vegetations, or severe congenital developmental abnormalities. The functional testing focuses on the following indicators: leaflet number, leaflet mobility, annular diameter, and regurgitation or stenosis, to determine suitability for single valve root acquisition, double valve root acquisition, or domino donor application. This invention does not impose specific age restrictions on the donor, but donors from neonates to children are preferred to improve the match with pediatric recipients in terms of anatomical size and growth potential.

[0018] In this invention, the donor heart is preferably sourced from brain-dead donor hearts, circulatory-dead donor hearts, domino donor hearts, and donor hearts unsuitable for whole-heart transplantation but with intact and functional valves, preferably donor hearts unsuitable for whole-heart transplantation but with intact and functional valves, to improve material utilization. This invention does not impose any particular limitations on the method of obtaining and separating the donor heart; methods well-known in the art can be used, such as performing a heart removal surgery after the doctor confirms the donor's brain death, with the entire process conducted under sterile conditions. The removed donor heart is immediately perfused with a pre-cooled preservation solution to thoroughly remove residual blood and reduce tissue metabolic levels. The pre-cooling temperature is preferably 0-4°C, and the perfusion volume is preferably 20-500 mL, which can be 50-200 mL. The preservation solution preferably includes any one of the following: University of Wisconsin preservation solution, HTK solution, and Celsior solution. After the heart is emptied through perfusion, the superior and inferior vena cava, pulmonary veins, ascending aorta, and main pulmonary artery are sequentially disconnected to obtain the donor heart intact. Preferably, the total thermal ischemia time from the determination of death to the removal of the donor heart should not exceed 10 minutes, more preferably not exceed 5 minutes, and the entire operation should be carried out under low temperature conditions on ice to preserve the viability of valve tissue cells to the greatest extent possible, so as to minimize thermal ischemia damage.

[0019] In this invention, the target valve tissue preferably includes at least one of the following structures: aortic root, pulmonary artery root, and double valve root structure. The harvesting is performed using a complete valve root unit harvesting method, rather than simply harvesting the valve leaflets, to maximize the preservation of the natural valve geometry, cell viability, and subsequent growth potential. The harvested target valve tissue preferably includes the complete valve leaflet, complete valve annulus, at least one complete valve sinus, proximal continuous ventricular myocardial border or annulus fibrosus tissue, and distal continuous ascending aortic wall or aortopulmonary wall tissue. The proximal tissue retention length of the target valve tissue is preferably 1-5 mm, and can be 2-3 mm, to facilitate subsequent suturing and fixation. The distal vessel wall retention length of the target valve tissue is preferably 3-20 mm, and can be 5-10 mm, to meet the outflow tract reconstruction and growth adaptation needs of pediatric recipients. For aortic root grafts, the excised donor heart is first placed on a sterile ice-covered operating table to fully expose the ascending aorta and aortic root. Observe the sinus duct junction longitudinally along the ascending aorta. Make a circumferential incision in the ascending aortic wall approximately 5-15 mm above the sinus duct junction as a distal incision, preserving the proximal aortic wall intact. Then, dissect proximally along the outer wall of the aorta to expose the entire aortic sinus structure, taking care to avoid damaging the coronary artery ostia. Make a circumferential incision around the left ventricular outflow tract approximately 2-3 mm below the aortic valve annulus, close to the lower edge of the annulus, forming a stable proximal suture ring. During the incision, cut slowly along the fibrous direction of the valve annulus to avoid damaging the leaflet attachment, free edge of the leaflets, and occlusal area. After completing the circumferential incision, remove the intact aortic root along with the tricuspid aortic valve, the entire aortic sinus, the valve annulus, the proximal myocardial border, and the distal ascending aortic wall to form a complete aortic root graft. For pulmonary artery root grafts, the main pulmonary artery and right ventricular outflow tract region are first exposed. Starting distal to the main pulmonary artery trunk, a transverse incision is made proximal to or before the bifurcation, preserving the main pulmonary artery trunk intact as the distal anastomosis segment. Subsequently, the pulmonary artery is dissected proximally along its outer wall to fully expose the pulmonary valve sinuses and annulus. A circular incision is made below the pulmonary valve annulus along the right ventricular outflow tract myocardial sleeve, maintaining a continuous and intact incision. The incision direction is progressively from the anterior wall to the posterior wall, slowly cutting along the natural course of the valve annulus to avoid damaging the pulmonary valve leaflet attachment margins and valve sinus structures. After completion, the intact pulmonary artery root, along with the pulmonary valve tricuspid structure, intact annulus, all pulmonary sinuses, proximal right ventricular outflow tract myocardial sleeve, and distal main pulmonary artery trunk, is removed as a whole to form a complete pulmonary artery root graft. When harvesting dual valve roots, it is preferable to complete the aortic root dissection first, followed by pulmonary root dissection. First, the aortic root is cut using the method described above. After cutting the proximal and distal ends of the aortic root, the continuity of the tissue between the aortic root and the pulmonary artery root is preserved to avoid direct traction. Then, sharp dissection is performed along the septum between the aortic root and the pulmonary artery root to gradually expose the outer wall of the pulmonary artery root. Finally, the pulmonary artery root is harvested using the same method as the pulmonary artery root cutting procedure.During the dissection process, special care should be taken to avoid damaging the posterior wall of the aortic root, the anterior wall of the pulmonary artery root, and the shared ventricular septal myometrial margin between the two. If necessary, adjacent ventricular septal myometrial margin or vessel wall tissue can be preserved as living tissue material for simultaneous ventricular septal defect repair, right ventricular outflow tract reconstruction, or pulmonary artery patch formation. Throughout the harvesting process, the tissue should be kept moist and cold, avoiding mechanical traction, leaflet compression, and repeated flipping to maximize the preservation of valvular endothelial cell viability, valvular interstitial cell integrity, and natural extracellular matrix structure, thereby ensuring long-term functional stability and continuous growth capacity of the graft after implantation. The optimal harvesting time for the target intact valve tissue is within 30 minutes to maximize cell viability.

[0020] In this invention, the harvested intact valve tissue immediately enters a low-temperature viability preservation stage. The intact valve tissue is placed in a sterile, sealed container and immersed in a pre-cooled preservation solution. The temperature is controlled throughout the process at 0–8°C, preferably 2–6°C, and most preferably 4°C. The preservation time can be controlled within the range of 0.5–48 h, specifically 2–24 h, or even 12 h, depending on donor transportation and surgical arrangements. During this process, decellularization, chemical cross-linking fixation, cryo-deactivation, or other treatments that may damage cell viability are avoided to preserve the natural integrity and subsequent growth potential of the valve endothelial cells, valve interstitial cells, and extracellular matrix as much as possible.

[0021] Before implantation, the donor-recipient match is comprehensively assessed based on the recipient's age, weight, body surface area (BSA), valve annulus diameter, outflow tract anatomy, and Z-score. Individualized trimming and shaping of the target intact valve tissue is then performed, including adjusting the proximal suture edge length, trimming the distal vessel wall length, removing redundant myocardial tissue, and retaining necessary patch tissue to ensure a better fit between the graft and the recipient's anatomy. The focus is on ensuring the graft meets current hemodynamic requirements while preserving potential for future growth. During proximal trimming, a continuous and intact valve annulus suture is preserved, and redundant myocardial tissue is removed to avoid post-suture annulus deformation, local stenosis, or excessive anastomotic tension. At the aortic root, the focus is on maintaining the continuity of the left ventricular outflow tract, and at the pulmonary artery root, the focus is on ensuring the patency of the right ventricular outflow tract. During distal trimming, the vessel wall length is adjusted according to the recipient's outflow tract length to avoid both excessively short lengths leading to excessive anastomotic tension and excessively long lengths causing vessel torsion, folding, or local blood flow disturbances. For neonates and infants, it is preferable to retain a certain length of growth-retaining tissue to accommodate subsequent physical development. For patients with ventricular septal defects, right ventricular outflow tract stenosis, pulmonary artery dysplasia, or complex outflow tract malformations, donor-derived muscular or vascular patches can be preserved simultaneously for concurrent anatomical reconstruction to further improve the overall growth potential and long-term durability of the graft. The optimal range for the diameter of the pediatric valve annulus is 5–25 mm, more preferably 6–18 mm.

[0022] In this invention, the biocompatible allogeneic valve preferably further includes pre-implantation quality assessment. The pre-implantation quality assessment method preferably includes at least one of the following: appearance integrity check, leaflet mobility assessment, occlusion integrity test, and sterility test.

[0023] In this invention, the prepared living allogeneic valve graft preferably includes the following structures: leaflet tissue, annular tissue, sinus tissue, proximal support tissue, distal vessel wall tissue, and optional patch tissue. The leaflet tissue includes complete semilunar leaflets, preferably aortic or pulmonary leaflets, and preferably a tricuspid structure, but may also include bicuspid or abnormal variations depending on the donor's actual anatomy. The annular tissue preferably includes a complete annular structure continuous with the leaflet base, used to maintain the mechanical stability of the graft after implantation and subsequent annular diameter expansion as the recipient grows. The sinus tissue preferably includes at least one complete sinus, preferably retaining all sinus structures to maintain normal vortex formation and leaflet opening and closing dynamics. The proximal support tissue preferably includes a continuous portion of the ventricular myocardial border or outflow tract fibrous annulus tissue proximal to the annulus, preferably retaining a continuous tissue edge of 1-5 mm, which can be 2-3 mm, for anastomosis and structural support. The distal vessel wall tissue preferably includes continuous tissue of the ascending aortic wall or the main pulmonary artery wall, preferably with a length of 3-20 mm, but can be 5-10 mm, for distal anastomosis and subsequent growth adaptation in children. Optional patch tissue preferably includes donor-derived vessel wall patches or muscular tissue patches for simultaneous repair of ventricular septal defects, outflow tract reconstruction, or pulmonary artery branching. The living allogeneic valve graft preferably meets the following preferred parameters: valve tissue cell viability is preferably ≥85%, most preferably ≥95%. The method for determining valve tissue cell viability is to dissociate waste tissue obtained during the trimming process into single cells, stain with AOPI or trypan blue, determine the number of viable cells, and calculate the percentage of viable cells out of the total cell count. The cold ischemia time (total cold ischemia time from donor removal to recipient implantation) is controlled within 0.5-48 h, preferably 1-24 h, most preferably 2-12 h. The storage temperature is preferably maintained at 0-8℃ throughout the entire process, preferably 2-6℃, most preferably 4℃. The valve annulus diameter matching range is suitable for children with a valve annulus diameter of 5~25 mm, preferably 6~18 mm.

[0024] The present invention also provides the application of the growable living allogeneic valve prepared by the above preparation method in the preparation of biomaterials for treating congenital heart valve diseases in children.

[0025] In this invention, the method for treating congenital valvular heart disease in children preferably includes surgical replacement of the aortic valve and / or pulmonary valve. The biomaterial includes biomedical materials.

[0026] The following detailed description, in conjunction with embodiments, illustrates a method for preparing a growable living allogeneic valve for pediatric valve replacement and its application, but these descriptions should not be construed as limiting the scope of protection of this invention.

[0027] Example 1 A method for preparing a growable living allogeneic valve for pediatric valve replacement Step 1: Donor screening and source determination Allogeneic donor hearts are selected as the source of transplantation material. Donor sources can include brain-dead donors, circulating-dead donors (DCD), domino donor hearts, and donor hearts that are not suitable for whole-heart transplantation but have intact valve structure and normal function. The donor heart must be confirmed by preoperative ultrasound or ex vivo gross examination to have an intact semilunar valve structure, good leaflet mobility, and no obvious calcification, tearing, infectious vegetations, or congenital developmental abnormalities. Preoperative evaluation of the donor is performed. Echocardiography and ex vivo gross examination confirm that the donor aortic or pulmonary valve structure is intact, the leaflet opening and closing function is good, and there is no obvious calcification, leaflet tearing, infectious vegetations, or severe congenital developmental abnormalities. The number of leaflets, leaflet mobility, annular diameter, and regurgitation or stenosis are specifically assessed to determine suitability for single valve root harvesting, double valve root harvesting, or domino donor application.

[0028] Step 2: Donor Heart Acquisition Immediately after confirming brain death in the donor, a median sternotomy is performed to open the pericardium and fully expose the ascending aorta, pulmonary artery, and major cardiac and vascular structures. Aortic ligation is completed within 5 minutes of confirmation of death, and pre-cooled preservation fluid is infused through the aortic root to rapidly stop the heart and reduce tissue metabolism. The preservation fluid can be HTK solution, with an infusion temperature of 0–4°C and an infusion volume of 50–200 mL. After cardiac evacuation, the superior and inferior vena cava, pulmonary veins, ascending aorta, and pulmonary artery are sequentially disconnected to obtain the donor heart intact. The total warm ischemia time from confirmation of death to removal of the donor heart is controlled to not exceed 10 minutes, and the entire procedure is performed under cryogenic conditions on ice to maximize the preservation of valvular tissue cell viability.

[0029] Step 3: Harvesting the target valve tissue The aortic root, pulmonary artery root, or double valve root are selected as grafts based on the recipient's clinical needs. All harvesting is performed using a complete valve root unit approach, rather than simply harvesting the valve leaflets, to maximize the preservation of the natural valve geometry, cell viability, and subsequent growth potential. The harvested area includes the complete valve leaflet, complete valve annulus, all valve sinuses, sinoduct commissure, proximal continuous ventricular myocardial border, and distal continuous vascular wall structure.

[0030] For aortic root grafts, the excised donor heart is first placed on a sterile ice-covered operating table to fully expose the ascending aorta and aortic root. The sinus duct junction is observed longitudinally along the ascending aorta. A circumferential incision is made in the ascending aortic wall approximately 5-15 mm above the sinus duct junction as a distal incision, preserving the proximal aortic wall intact. The entire aortic sinus structure is then dissected proximally along the outer wall of the aorta, taking care to avoid damaging the coronary artery ostia. A circumferential incision is made approximately 2-3 mm below the aortic valve annulus, around the left ventricular outflow tract, close to the lower edge of the annulus to form a stable proximal suture ring. During the incision, the incision should be made slowly along the fibrous direction of the valve annulus to avoid damaging the leaflet attachment, free edge of the leaflets, and occlusal region. After completing the circumferential incision, the intact aortic root, along with the tricuspid aortic valve, the entire aortic sinus, the valve annulus, the proximal myocardial border, and the distal ascending aortic wall, is removed as a whole, forming a complete aortic root graft.

[0031] For pulmonary artery root grafts, the main pulmonary artery and right ventricular outflow tract region are first exposed. Starting distal to the main pulmonary artery trunk, a transverse incision is made proximal to or before the bifurcation, preserving the main pulmonary artery trunk intact as the distal anastomosis segment. Subsequently, the pulmonary artery is dissected proximally along its outer wall to fully expose the pulmonary valve sinuses and annulus. A circular incision is made below the pulmonary valve annulus along the right ventricular outflow tract myocardial sleeve, maintaining a continuous and intact incision. The incision direction is progressively from the anterior wall to the posterior wall, slowly cutting along the natural course of the valve annulus to avoid damaging the pulmonary valve leaflet attachment margins and valve sinus structures. After completion, the intact pulmonary artery root, along with the pulmonary valve tricuspid structure, intact annulus, all pulmonary sinuses, proximal right ventricular outflow tract myocardial sleeve, and distal main pulmonary artery trunk, is removed as a whole to form a complete pulmonary artery root graft.

[0032] When harvesting the pulmonary artery root, it is preferable to first separate the aortic root and then free the pulmonary artery root. First, the aortic root is harvested using the method described above. After incising the proximal and distal ends of the aortic root, the continuity of the tissue between the aortic and pulmonary artery roots is preserved to avoid direct, forceful traction. Then, sharp dissection is performed along the septum between the aortic and pulmonary artery roots to gradually expose the outer wall of the pulmonary artery root. The pulmonary artery root is then harvested using the same method. During the dissection process, care should be taken to avoid damaging the posterior wall of the aortic root, the anterior wall of the pulmonary artery root, and the shared ventricular septal margin tissue between them. If necessary, adjacent ventricular septal margin or vessel wall tissue can be preserved as living tissue material for simultaneous ventricular septal defect repair, right ventricular outflow tract reconstruction, or pulmonary artery patch formation.

[0033] Throughout the harvesting process, the tissue should be kept moist and at a low temperature. Mechanical traction, leaflet compression, and repeated flipping should be avoided to maximize the preservation of endothelial cell activity, interstitial cell integrity, and natural extracellular matrix structure, thereby ensuring long-term functional stability and continuous growth capacity of the graft after implantation.

[0034] Step 4: Low-temperature activity protection The harvested live allogeneic valve tissue immediately enters a cryogenic viability preservation phase. The graft is placed in a sterile, sealed container and immersed in a pre-cooled preservation solution, with the temperature maintained at 4°C throughout the process. The preservation time can be controlled within 12 hours depending on donor transportation and surgical arrangements. During this process, decellularization, chemical cross-linking fixation, cryoablation, or other treatments that may damage cell viability are avoided to preserve the natural integrity and subsequent growth potential of the valve endothelial cells, valve interstitial cells, and extracellular matrix as much as possible.

[0035] Step 5: Individualized shaping Donor-recipient matching is comprehensively assessed based on the recipient's age, weight, body surface area (BSA), valve annulus diameter, and Z-score. The focus is on ensuring the graft meets current hemodynamic requirements while preserving future growth redundancy. During proximal trimming, a continuous and intact valve annulus suture ring is preserved, and redundant myocardial tissue is removed to avoid post-suture annulus deformation, local stenosis, or excessive anastomotic tension. At the aortic root, the focus is on maintaining left ventricular outflow tract continuity, and at the pulmonary artery root, the focus is on ensuring right ventricular outflow tract patency. During distal trimming, the vessel wall length is adjusted according to the recipient's outflow tract length to avoid both excessively short sections leading to excessive anastomotic tension and excessively long sections causing vessel torsion, folding, or local blood flow disturbances. For neonates and infants, preserving a certain length of growth redundancy is preferred to accommodate subsequent physical development. For patients with ventricular septal defects, right ventricular outflow tract stenosis, pulmonary artery dysplasia, or complex outflow tract malformations, donor-derived muscular or vascular patches can be preserved simultaneously for anatomical reconstruction to further enhance the overall growth potential and long-term durability of the graft. The suitable pediatric valve annulus diameter ranges from 6 to 18 mm.

[0036] Step 6: Pre-implantation quality assessment Prior to implantation, the prepared living allogeneic valve graft undergoes a quality assessment, including inspection of its appearance integrity, leaflet mobility, occlusion integrity, and sterility. Only qualified hearts are used for surgery.

[0037] The prepared live allogeneic valve comprises the following structural components: 1) The leaflet tissue includes complete aortic valve leaflets or pulmonary valve leaflets, and the number of leaflets is trilobal, bilobal, or abnormally varied.

[0038] 2) The valve annulus tissue includes a complete valve annulus structure that is continuous with the base of the valve leaflet, which is used to maintain the mechanical stability of the graft after implantation and to allow the annulus diameter to expand as the recipient grows.

[0039] 3) The valve sinus tissue includes at least one complete valve sinus to maintain normal vortex formation and leaflet opening and closing dynamics.

[0040] 4) Proximal supporting tissue includes a continuous portion of the ventricular myocardial border or outflow tract fibrous annulus tissue proximal to the valve annulus, retaining a 3mm continuous tissue edge for anastomosis and structural support.

[0041] 5) The distal vascular wall tissue, including continuous tissue of the ascending aortic wall or the main pulmonary artery wall, is preserved in a length of 5 mm for distal anastomosis and subsequent growth adaptation in children.

[0042] 6) Optional patch tissues include donor-derived vascular wall patches or muscular tissue patches for simultaneous repair of ventricular septal defects, outflow tract reconstruction, or pulmonary artery branching.

[0043] Example 2 The method for preparing a growable living allogeneic valve for pediatric valve replacement was validated through clinical cases. A total of 3 clinical application cases were implemented, including 2 cases for pulmonary valve replacement and 1 case for aortic valve replacement.

[0044] According to the preparation process described in this invention, suitable allogeneic donor hearts were selected. Preoperative ultrasound confirmed that the donor valve structure was intact, its opening and closing function was good, and there was no obvious calcification, tearing, or vegetation. Immediately after the donor heart was removed from the body, it was cryopreserved with a pre-cooled preservation solution, and the target valve tissue was harvested under aseptic conditions. In two cases, the pulmonary artery root was harvested as the pulmonary valve graft, and in one case, the aortic root was harvested as the aortic valve graft. All grafts retained the leaflets, annulus, valve sinus, and continuous proximal and distal vascular wall structures intact, and were individually trimmed according to the recipient's anatomical dimensions before implantation.

[0045] like Figure 1 As shown, the diameter of the transplanted valves in all three patients continued to increase with follow-up time. Immediately after surgery, echocardiography showed that the average diameter of the transplanted valves in the three recipients was 9.2 mm. After 26 months of follow-up, repeat echocardiography showed that the average valve diameter had increased to 15.7 mm, an increase of 6.5 mm compared to immediately after surgery, representing an increase of approximately 70.7%.

[0046] The results showed that the living allogeneic valve grafts prepared in this invention maintained good structural integrity and functional stability in pediatric recipients and could continuously expand as the recipients grew, confirming their significant growth potential. Among them, no significant valvular stenosis, leaflet calcification, or severe regurgitation was observed in two pulmonary valve grafts and one aortic valve graft during the follow-up period, suggesting that the preparation method of this invention has good clinical feasibility and long-term application prospects.

[0047] Comparative Example 1 Case 1: A living allogeneic valve was prepared according to the method in Example 1, with the following differences: the total warm ischemia time from the determination of death to the removal of the donor heart was 15 minutes, which exceeded 10 minutes; the time from the removal of the donor heart to the acquisition of the living allogeneic valve graft was 40 minutes, which exceeded 30 minutes. Cell viability was measured.

[0048] The measured cell viability was 80%, below 85%. This indicates that the time between the donor heart's removal from the donor and the preparation time directly affect cell viability. Therefore, by strictly controlling the time according to the method of this invention, the viability of most cells reached ≥85%.

[0049] Case 2: When the valve was not obtained according to the method in Example 1, the valve was not completely removed, resulting in damage to the valve annulus at the valve root (see Example 1). Figure 2 During implantation, insufficient space at the anastomosis site may cause incomplete closure of the valve annulus due to traction. Therefore, it may be discarded because it does not meet the criteria for implantation in the body.

[0050] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a living, allogeneic valve, characterized in that, Includes the following steps: After rinsing the isolated donor heart with pre-cooled preservation solution, the target intact valve tissue was excised under aseptic conditions and then placed in a sterile environment at 0~8℃ for low-temperature viability protection. Before the implantation surgery, the target intact valve tissue is individually trimmed and shaped according to the child recipient's age, weight, valve annulus diameter and outflow tract anatomy, to obtain a living allogeneic valve that can grow.

2. The preparation method according to claim 1, characterized in that, The target valve tissue includes at least one of the following structures: aortic root, pulmonary artery root, and double valve root structure.

3. The preparation method according to claim 1 or 2, characterized in that, The scope of the target valve tissue to be harvested includes the complete valve leaflet, complete valve annulus, at least one complete valve sinus, continuous proximal ventricular myocardial border or annulus fibrosus tissue, and continuous distal ascending aortic wall or aortopulmonary artery wall tissue.

4. The preparation method according to claim 3, characterized in that, The proximal tissue retention length of the target valve tissue is 1~5mm; The distal vessel wall of the target valve tissue is preserved for a length of 3-20 mm.

5. The preparation method according to claim 1, characterized in that, The sources of the donor hearts include brain-dead donor hearts, circulatory-dead donor hearts, domino donor hearts, and donor hearts that are not suitable for whole-heart transplantation but have intact valve structure and normal function.

6. The preparation method according to claim 5, characterized in that, The donor heart also includes functional testing; The functional tests include echocardiography and / or in vitro visual examination to confirm whether the semilunar valve structure is intact, whether the leaflets move well, and whether there is obvious calcification, tearing, infectious vegetations, or congenital developmental abnormalities.

7. The preparation method according to claim 1, characterized in that, The time for the donor heart to leave the body is controlled within 30 minutes.

8. The preparation method according to claim 1, characterized in that, The preservation solution includes any one of the following: University of Wisconsin preservation solution, HTK solution, and Celsior solution.

9. The preparation method according to claim 1, characterized in that, The growable biocompatible valve also includes pre-implantation quality assessment. The preimplantation quality assessment method includes at least one of the following: appearance integrity inspection, leaflet mobility assessment, occlusion integrity test, and sterility test.

10. The use of the growable living allogeneic valve prepared by the preparation method according to any one of claims 1 to 9 in the preparation of biomaterials for treating congenital heart valve diseases in children.

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