CN116942914A - Human allogenic decellularized matrix tissue engineering heart valve, preparation method and application - Google Patents
Human allogenic decellularized matrix tissue engineering heart valve, preparation method and application Download PDFInfo
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3895—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
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- D—TEXTILES; PAPER
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- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/20—Materials or treatment for tissue regeneration for reconstruction of the heart, e.g. heart valves
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Abstract
The invention discloses a heart valve of human allogenic decellularized matrix tissue engineering, a preparation method and application thereof, which comprises the steps of preparing a composite tubular bracket and/or a composite film of polyglycolic acid and polyethylene glycol polymer by adopting electrostatic spinning, planting seed cells on the composite tubular bracket and/or the composite film, simulating the perfusion culture of arterial pulsating flow pressure and axial cyclic stretching tension of a human body under a first preset culture condition, obtaining tubular and/or sheet-shaped engineering tissue composed of the seed cells and extracellular matrix, removing tube wall cells from the tubular and/or sheet-shaped engineering tissue, obtaining tubular engineering tissue and/or sheet-shaped engineering film composed of extracellular matrix, cutting the tubular engineering tissue and/or sheet-shaped engineering film, and preparing the decellularized matrix tissue engineering heart valve leaflet material by utilizing the leaflet material into the heart valve of the decellularized matrix tissue engineering including heart valve of conventional surgical operation and minimally invasive heart valve.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a heart valve of a human allogeneic decellularized matrix tissue engineering, a preparation method and application thereof.
Background
Heart valves, including mitral valve, tricuspid valve, aortic valve, and pulmonary valve, are important structures to ensure unidirectional blood flow. The heart valve is opened and closed more than 10 ten thousand times per day, for a total of about 4000 ten thousand times per year. When heart valves are affected by congenital malformations or acquired factors, such as degenerative changes, ischemic necrosis, infection or trauma, blood flow and normal heart function are affected, such as by valvular heart disease. According to the related investigation, about 2500 thousands of valvular heart disease patients are domestically weighted with a prevalence of 3.8%.
At present, the most common treatment method for the valvular heart disease is surgical heart valve replacement, 25-30 ten thousand valve replacement operations are about 25-30 ten thousand times a year, the trend is obviously growing in China, about 8 ten thousand times a year, and mechanical valve or biological valve prosthesis and minimally invasive intervention valve replacement are adopted at present. The service life of the mechanical valve can be longer than 50 years, but patients need to receive anticoagulation treatment for life, so bleeding and thrombus complications are caused, and the life is endangered when serious; biological valves, including bovine and porcine heart valves, have limited life and the human cells cannot reconstruct the structural tissue of the valve leaflets and need to be operated again after decay.
In addition, the use of cryopreserved technical human allogeneic heart valves has been commercialized for nearly forty years, and the valves have superior performance, do not require lifelong anticoagulation, have a lifetime comparable to mechanical valves, and can be used for lifetime without re-surgery. But because of limited sources of donated heart valves, such products are particularly blank in our country. At present, a new generation of high molecular material heart valves are being developed globally, such as Tria valve of Foldax company in the United states, domestic PeJia medical TaurusApex valve and the like, but the high molecular material valves still need anticoagulation treatment, and the biocompatibility is obviously inferior to that of biological valves. In recent years, tissue engineering technology which is widely paid attention to has been successfully applied to artificial blood vessel development and production, wherein human allogenic small-caliber tissue engineering blood vessels are about to be commercialized, the wall structure of the tissue engineering blood vessels is mainly human blood vessel wall I, III type collagen, and the tissue engineering blood vessels have proved to have excellent biocompatibility and mechanical properties. The heart valve with the human allogenic tissue engineering is cultured and developed in vitro by using the tissue engineering technology, and the valve leaflet structure has the advantages of rapid endothelialization, self-repair and reconstruction in vivo, good tissue compatibility, no immunogenicity, good durability, no need of anticoagulation treatment and the like, and represents the development direction of heart valves of the next generation.
Disclosure of Invention
Accordingly, the present invention provides a heart valve with human allogeneic decellularized matrix tissue engineering, a preparation method and application thereof, so as to solve or partially solve the above problems.
In order to achieve the above object, the present invention also provides a method for preparing a heart valve of human allogenic decellularized matrix tissue engineering, comprising:
preparing a composite tubular bracket and/or a composite film of polyglycolic acid and polyethylene glycol polymer by adopting electrostatic spinning;
planting seed cells in a composite tubular bracket and/or a composite film, and simulating the arterial pulsating pressure and the axial cyclic stretching tension of an organism under a first preset culture condition to perform perfusion culture to obtain tubular and/or sheet engineering tissue consisting of the seed cells and extracellular matrix, wherein the axial cyclic stretching tension condition in the first preset culture condition comprises: firstly, statically culturing for 1-10 days, then applying cyclic stretching tension in the axial direction to culture for 6-10 days under the condition of 1-3% of axial deformation, then raising the axial stretching deformation to 5% -7% to continue culturing, and raising the axial stretching deformation to 9% -10% again in the last week of the culturing process;
removing cells from the tubular and/or sheet-like engineering tissue to remove cells from the tubular wall, thereby obtaining a tubular-like engineering tissue and/or sheet-like engineering membrane composed of extracellular matrix;
cutting the tubular engineering tissue and/or the sheet engineering membrane to obtain a valve leaflet material of the acellular matrix tissue engineering heart valve, and preparing the acellular matrix tissue engineering heart valve by using the valve leaflet material, wherein the acellular matrix tissue engineering heart valve comprises a conventional surgical heart valve and a minimally invasive intervention heart valve.
Preferably, in the method for preparing the heart valve of the human allogeneic decellularized matrix tissue engineering, the step of planting the seed cells in a composite tubular stent and/or a composite film, and performing perfusion culture under the condition of simulating the pulsating flow pressure and the axial cyclic stretching tension of the body artery under the first preset culture condition to obtain tubular and/or sheet engineering tissue consisting of the seed cells and extracellular matrix specifically comprises the following steps:
the seed cells are planted in the composite tubular bracket and/or the composite film, and the tubular and/or sheet engineering tissue consisting of the seed cells and extracellular matrix is obtained by simulating the arterial pulsating flow pressure and the axial cyclic stretching tension of the organism under the first preset culture condition and carrying out perfusion culture for 4-12 weeks.
Preferably, in the method for preparing the heart valve of the human allogeneic decellularized matrix tissue engineering, the step of planting seed cells on a composite tubular stent and/or a composite film, and performing perfusion culture under conditions simulating arterial pulsating flow pressure and axial cyclic stretching tension of an organism under a first preset culture condition to obtain tubular and/or sheet-shaped engineering tissue composed of the seed cells and extracellular matrix, wherein the first preset culture condition simulates arterial pulsating flow pressure culture conditions of the organism including:
firstly, static culturing for 1-10 days, then culturing for 6-10 days under arterial pulse flow with pressure of 0-90mmHg, then raising arterial pulse flow to 90-130mmHg, continuously culturing, and raising arterial pulse flow pressure to 130-180mmHg in the last week of the culturing process.
Preferably, in the preparation method of the human allogeneic decellularized matrix tissue engineering heart valve, the inner layer of the composite tubular stent and/or the composite film is polyglycolic acid, the outer layer is polyethylene glycol polymer, and the thickness ratio of the polyglycolic acid of the inner layer to the polyethylene glycol polymer of the outer layer is 1:1-5:1.
Preferably, in the method for preparing the heart valve of the human allogeneic decellularized matrix tissue engineering, the step of preparing the composite tubular stent and/or the composite film of polyglycolic acid and polyethylene glycol polymer by adopting electrostatic spinning specifically comprises the following steps:
preparing a composite tubular bracket of polyglycolic acid and polyethylene glycol polymer by adopting electrostatic spinning, wherein the inner diameter of the composite tubular bracket is 18-30mm, the total thickness of the pipe wall is 0.2-2mm, the length is 2-10cm, the porosity of the pipe wall of the bracket is 80-95%, and the aperture is 50nm-200um;
or the steps for preparing the composite tubular stent and/or the composite film of polyglycolic acid and polyethylene glycol polymer by adopting electrostatic spinning are specifically as follows:
and preparing a composite film of polyglycolic acid and polyethylene glycol polymer by adopting electrostatic spinning, wherein the total thickness of the composite film is 0.5-2mm, the length is 2-10cm, the width is 6-9 cm, the porosity of the bracket is 80-95%, and the aperture is 50nm-200 mu m.
Preferably, in the method for preparing the human allogenic decellularized matrix tissue engineering heart valve, the step of removing the tubular and/or sheet engineering tissue to remove the tubular wall cells and obtaining the tubular engineering tissue and/or sheet engineering membrane composed of extracellular matrix specifically comprises the following steps:
placing the tubular and/or sheet-shaped engineering tissue into a configured decellularized reagent, and treating for 1-3h at room temperature; changing the decellularized reagent once every 1-3 hours for 3-6 times to obtain tubular engineering tissue and/or sheet engineering membrane composed of extracellular matrix; the decellularized reagent is prepared from 3- [ (3-cholesteryl amidopropyl) -diethylamine ] -propane sulfonic acid, disodium ethylenediamine tetraacetate (EDTA-2 Na), naCl, naOH and sterile deionized water.
Preferably, in the method for preparing the human allogeneic decellularized matrix tissue engineering heart valve, the seed cells include one or more of fibroblasts of adult human or mammal, valve mesenchymal cells, mesenchymal stem cells and fibroblasts induced to differentiate from multifunctional stem cells, and valve mesenchymal cells.
Preferably, in the method of preparing the human allogeneic decellularized matrix tissue-engineered heart valve, the seed cells are derived from a single or multiple donors or cell banks using primary-10 generation cells.
In order to achieve the above object, the present invention also provides a heart valve of human allogeneic decellularized matrix tissue engineering, which is prepared by the above preparation method of the heart valve of human allogeneic decellularized matrix tissue engineering.
In order to achieve the above object, the present invention also provides a heart valve of a human allogenic decellularized matrix tissue engineering, which is composed of extracellular matrix components secreted by seed cells, and the extracellular matrix components have in-situ in vivo decellularization.
In order to achieve the above object, the present invention also provides an application of the human allogenic decellularized matrix tissue engineering heart valve, which is applied to a conventional surgical heart valve replacement or interventional heart valve replacement of a mitral valve, a tricuspid valve, an aortic valve and a pulmonary valve after being combined with a valve stent.
In order to achieve the above purpose, the invention also provides an application of the human allogeneic decellularized matrix tissue engineering heart valve, which is combined with a valve support and then applied to surgical replacement or interventional replacement, wherein the human allogeneic decellularized matrix tissue engineering heart valve can realize rapid endothelialization of the surface of a valve leaflet and decellularization of the tissue inside the valve leaflet after being implanted into a body due to the characteristic structure of the human allogeneic decellularized matrix tissue engineering heart valve, and reconstruct the new heart valve in situ in the body.
The invention has the following beneficial effects:
the invention provides a preparation method of a heart valve of a human allogeneic decellularized matrix tissue engineering, which comprises the steps of preparing a composite tubular bracket and/or a composite film of polyglycolic acid and polyethylene glycol polymer by adopting electrostatic spinning, planting seed cells on the composite tubular bracket and/or the composite film, and carrying out perfusion culture under the condition of simulating arterial pulsating flow pressure and axial cyclic stretching tension of an organism under a first preset culture condition to obtain tubular and/or sheet engineering tissue consisting of the seed cells and extracellular matrix, wherein the axial cyclic stretching tension condition in the first preset culture condition comprises: static culturing for 1-10 days, applying cyclic stretching tension in axial direction to make axial deformation 1-3%, culturing for 6-10 days, increasing axial stretching deformation to 5-7%, culturing, increasing axial stretching deformation to 9-10%, culturing, removing cell wall cells from tubular and/or sheet engineering tissue to obtain tubular engineering tissue and/or sheet engineering membrane composed of extracellular matrix, cutting to obtain heart valve material, the integrated or semi-integrated decellularized matrix tissue engineering heart valve material is combined with the bracket to form the decellularized matrix tissue engineering heart valve prosthesis, and after the decellularized matrix tissue engineering heart valve is matched with the bracket and implanted into a human body, the rapid endothelialization of the surface of the valve leaflet, the decellularization of the mesenchymal cells of the valve leaflet internal tissues can be realized, and the new heart valve is reconstructed in situ in vivo, so that the problems of long-term anticoagulation of the mechanical valve, immunogenicity and calcification of the biological valve are effectively solved.
Furthermore, the heart valve with the human allogenic decellularized matrix tissue has a special bionic extracellular matrix 3D structure, so that the heart valve is beneficial to cell adhesion and growth after being implanted into a body, thrombus is prevented from being formed, and meanwhile, the circulating progenitor cells can be captured on the surface of the valve leaflet, so that the progenitor cells are promoted to directionally differentiate, and valve tissues are reconstructed.
Furthermore, the human allogeneic decellularized matrix tissue engineering heart valve has good regeneration performance, and can solve the problem that the mechanical valve needs long-term anticoagulation, and the problems of immunogenicity and easy calcification possibly existing in the biological valve.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flowchart of one embodiment of a method of preparing an allogeneic decellularized matrix tissue-engineered heart valve of the invention;
FIG. 2 is a schematic view of one embodiment of the tubular decellularized matrix tissue engineering valve cutting of the present invention;
FIG. 3 is a schematic diagram of another embodiment of FIG. 2;
FIG. 4 is a schematic view of an embodiment of a process for preparing a surgical valve according to the present invention;
FIG. 5 is a schematic diagram of one embodiment of an application of the interventional valve of the present invention;
fig. 6 is a top view of the decellularized matrix tissue engineering heart valve prosthesis of fig. 5.
FIG. 7 is a schematic representation of in situ reconstruction of a new heart valve in vivo after implantation of the decellularized matrix tissue engineering valve in vivo.
The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.
Detailed Description
In the embodiment of the invention, the term "and/or" describes the association relation of the association objects, which means that three relations can exist, for example, a and/or B can be expressed as follows: a exists alone, A and B exist together, and B exists alone. The character "/" generally indicates that the context-dependent object is an "or" relationship.
It should be noted that the terms "first," "second," and the like in the description and the claims of the present invention and the above figures are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order.
The term "plurality" in embodiments of the present invention means two or more, and other adjectives are similar.
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the embodiments of the present invention will be described in detail below with reference to the accompanying drawings. However, it will be understood by those of ordinary skill in the art that in various embodiments of the present invention, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, the claimed technical solution of the present invention can be realized without these technical details and various changes and modifications based on the following embodiments. The following embodiments are divided for convenience of description, and should not be construed as limiting the specific implementation of the present invention, and the embodiments can be mutually combined and referred to without contradiction.
The invention provides a preparation method of a human allogeneic decellularized matrix tissue engineering heart valve, referring to fig. 1, the preparation method of the human allogeneic decellularized matrix tissue engineering heart valve comprises the following steps:
step S100, preparing a composite tubular stent and/or a composite film of polyglycolic acid and polyethylene glycol polymer by adopting electrostatic spinning;
more specifically, the inner layer of the composite tubular stent and/or the composite film is polyglycolic acid, the outer layer is polyethylene glycol polymer, and the thickness ratio of the polyglycolic acid of the inner layer to the polyethylene glycol polymer of the outer layer is 1:1-5:1. In other embodiments, the thickness ratio of polyglycolic acid in the inner layer to polyethylene glycol polymer in the outer layer may also be 2:1, 3:1, or 4:1. Thus the degradation rate is better.
Preparing a polyglycolic acid and polyethylene glycol polymer composite tubular stent by electrostatic spinning, wherein the inner diameter of the composite tubular stent is 18-30mm (in the embodiment, the inner diameter can be 19mm, 20mm, 22mm, 24mm, 26mm or 28 mm), the total thickness of the tube wall is 0.2-2mm (in the embodiment, the total thickness of the tube wall is 0.4mm, 0.8mm, 1.0mm, 1.4mm or 1.8 mm), the length is 2-10cm (in the embodiment, the length is 4cm, 6cm or 8 cm), the porosity of the tube wall of the stent is 80-95% (in the embodiment, the porosity of the tube wall can be 82%, 84%, 86%, 88%, 90%, 92% or 94%), and the aperture is 50nm-200um (in the embodiment, the aperture is 100nm, 200nm, 500nm, 1um, 50um or 100 um);
or the steps for preparing the composite tubular stent and/or the composite film of polyglycolic acid and polyethylene glycol polymer by adopting electrostatic spinning are specifically as follows:
the composite film of polyglycolic acid and polyethylene glycol polymer is prepared by electrospinning, wherein the total thickness of the composite film is 0.5-2mm (in the embodiment, the total thickness of the composite film is 0.6mm, 0.7mm, 0.8mm, 0.9mm, 1.0mm, 1.2mm, 1.4mm, 1.6mm or 1.8 mm), the length is 2-10cm (in the embodiment, the length is 4cm, 6cm or 8 cm), the width is 6cm-9cm (in the embodiment, the width is 6.5cm, 7cm, 8cm or 8.5 cm), the stent porosity is 80-95% (in the embodiment, the stent porosity can also be 82%, 84%, 86%, 88%, 90%, 92% or 94%), and the pore diameter is 50nm-200um.
Step S200, seed cells are planted in a composite tubular bracket and/or a composite film, and perfusion culture is simulated under a first preset culture condition under arterial pulsating flow pressure and axial cyclic stretching tension conditions of an organism, so as to obtain tubular and/or sheet engineering tissue composed of the seed cells and extracellular matrixes, wherein the axial cyclic stretching tension conditions in the first preset culture condition comprise: firstly, static culturing for 1-10 days, then applying cyclic stretching tension in the axial direction to culture for 6-10 days under the condition of 1-3% of axial deformation, then raising the axial stretching deformation to 5% -7% to continue culturing, and raising the axial stretching deformation to 9% -10% again in the last week of the culturing process.
The number of days for static culture may be 2 days, 4 days, 6 days, or 8 days, and a steady state may be achieved. The axial deformation is preferably 1.5%, 2%, or 2.5%. The culture is preferably conducted for 7 days, 8 days, or 9 days under conditions of 1-3% axial deformation for 6-10 days. The axial stretching deformation is increased to 5% -7% and the axial stretching deformation is preferably 5.5%, 6% or 6.5% when the culture is continued.
Preferably, the axial tension set is increased again to 9% -10% at the last week of the culturing process, in particular 4 weeks-12 weeks (in this example, the axial tension set may also be 9.5%).
In specific implementation, the step S200 includes: the seed cells are planted in the composite tubular bracket and/or the composite film, and the tubular and/or sheet engineering tissue consisting of the seed cells and extracellular matrix is obtained by simulating the arterial pulsating flow pressure and the axial cyclic stretching tension of the organism under the first preset culture condition and carrying out perfusion culture for 4-12 weeks.
It should be noted that the tubular and/or sheet-like engineered tissue may be composed of seed cells and extracellular matrix.
Wherein, the first preset culture condition simulates the arterial pulsating flow pressure culture condition of the organism and comprises the following steps:
the culture is continued for 1 to 10 days (the days of static culture may be 2 days, 4 days, 6 days, or 8 days) and then for 6 to 10 days (the days of culture may be 7 days, 8 days, or 9 days) under arterial pulsations of 0 to 90mmHg (in this example, arterial pulsations of 20mmHg, 30mmHg, 40mmHg, 50mmHg, 60mmHg, 70mmHg, or 80 mmHg), and then the arterial pulsations are raised to 90 to 130mmHg (in this example, arterial pulsations of 95mmHg, 100mmHg, 105mmHg, or 110 mmHg), and the arterial pulsations are raised to 130 to 180mmHg (in this example, arterial pulsations of 130mmHg, 140mmHg, 150mmHg, 160 mmHg) for the last week of the culture.
The seed cells include, but are not limited to, one or more of fibroblasts, valve stromal cells, mesenchymal stem cells and fibroblasts derived from inducing differentiation of multifunctional stem cells, or valve stromal cells, which are adult humans or mammals.
The seed cells employ primary-10 generation cells using single or multiple donor or cell bank sources.
Step S300, the tubular and/or sheet-shaped engineering tissue is decellularized to remove the cell of the tube wall, and a tubular engineering tissue and/or sheet-shaped engineering membrane composed of extracellular matrix is obtained;
the step of the decellularizing method in the step of decellularizing the tubular and/or sheet-shaped engineering tissue to remove the cells of the tubular wall and obtaining the tubular-shaped engineering tissue and/or sheet-shaped engineering membrane composed of extracellular matrix, specifically comprises the following steps:
placing the tubular and/or sheet-shaped engineering tissue into a configured decellularized reagent, and treating for 1-3h at room temperature; changing the decellularized reagent once every 1-3 hours for 3-6 times to obtain tubular engineering tissue and/or sheet engineering membrane composed of extracellular matrix; the decellularized reagent is prepared from 3- [ (3-cholesteryl amidopropyl) -diethylamine ] -propane sulfonic acid, disodium ethylenediamine tetraacetate (EDTA-2 Na), naCl, naOH and sterile deionized water.
Step S400, cutting the tubular engineering tissue and/or the sheet engineering membrane to obtain a valve leaflet material of the acellular matrix tissue engineering heart valve, and preparing the acellular matrix tissue engineering heart valve by using the valve leaflet material, wherein the acellular matrix tissue engineering heart valve comprises a conventional surgical heart valve and a minimally invasive intervention heart valve.
More specifically, fig. 2 and 3 illustrate an embodiment of cutting the tubular tissue and/or sheet-like tissue engineering film, which is fixed inside the stent by stitching or bonding after cutting. Wherein the stent is a heart valve stent, and the material of the heart valve stent can be, but is not limited to, degradable material or non-degradable material.
The invention provides a preparation method of a heart valve of a human allogeneic decellularized matrix tissue engineering, which comprises the steps of preparing a composite tubular bracket and/or a composite film of polyglycolic acid and polyethylene glycol polymer by adopting electrostatic spinning, planting seed cells on the composite tubular bracket and/or the composite film, and carrying out perfusion culture under the condition of simulating arterial pulsating flow pressure and axial cyclic stretching tension of an organism under a first preset culture condition to obtain tubular and/or sheet engineering tissue consisting of the seed cells and extracellular matrix, wherein the axial cyclic stretching tension condition in the first preset culture condition comprises: static culturing for 1-10 days, applying cyclic stretching tension in axial direction to make axial deformation 1-3%, culturing for 6-10 days, increasing axial stretching deformation to 5-7%, culturing, increasing axial stretching deformation to 9-10%, culturing, removing cell wall cells from tubular and/or sheet engineering tissue to obtain tubular engineering tissue and/or sheet engineering membrane composed of extracellular matrix, cutting to obtain heart valve material, the integrated or semi-integrated decellularized matrix tissue engineering heart valve material is combined with the bracket to form the decellularized matrix tissue engineering heart valve prosthesis, and after the decellularized matrix tissue engineering heart valve is matched with the bracket and implanted into a human body, the rapid endothelialization of the surface of the valve leaflet, the decellularization of the mesenchymal cells of the valve leaflet internal tissues can be realized, and the in-vivo in-situ reconstruction of the new heart valve can effectively solve the problem that the mechanical valve needs long-term anticoagulation and the problems of immunogenicity and calcification of the biological valve.
In order to achieve the above purpose, the invention also provides a human allogenic decellularized matrix tissue engineering heart valve, which is prepared by adopting the preparation method of the human allogenic decellularized matrix tissue engineering heart valve.
The embodiment of the human allogenic decellularized matrix tissue engineering heart valve comprises the embodiment of the preparation method of the human allogenic decellularized matrix tissue engineering heart valve, and the beneficial effects of the preparation method of the human allogenic decellularized matrix tissue engineering heart valve can also be applied to the human allogenic decellularized matrix tissue engineering heart valve.
More specifically, the human allogeneic decellularized matrix tissue engineering heart valve is composed of extracellular matrix components secreted by seed cells, which components have in vivo in situ re-cytotoxicity.
More specifically, the extracellular matrix protein of the human allogeneic decellularized matrix tissue-engineered heart valve can include, but is not limited to, collagen i+collagen III, or collagen i+collagen iii+elastin.
In order to achieve the above object, the present invention also provides a heart valve of a human allogenic decellularized matrix tissue engineering, which is composed of extracellular matrix components secreted by seed cells, and the extracellular matrix components have in-situ in vivo decellularization.
More specifically, the extracellular matrix protein of the human allogeneic decellularized matrix tissue-engineered heart valve can include, but is not limited to, collagen i+collagen III, or collagen i+collagen iii+elastin.
Example 1 preparation method of human allogeneic Decellularized stromal tissue engineering heart valve
(1) Preparation of composite tubular stents and/or composite films
And preparing the composite tubular stent and/or the composite film of polyglycolic acid and polyethylene glycol polymer by adopting electrostatic spinning.
Specifically, polyglycolic acid and polyethylene glycol are respectively dissolved in an organic solvent to obtain uniform and stable spinning solution, electrostatic spinning is sequentially carried out, and a Teflon coated metal rod with the outer diameter of 18-30mm is used for receiving, so that an oriented fiber tubular composite tubular stent is obtained. Respectively dissolving polyglycolic acid and polyethylene glycol in an organic solvent to obtain uniform and stable spinning solution, sequentially carrying out electrostatic spinning, and receiving by using a Teflon coated metal plate with the length of 200mm and the width of 200mm to obtain the unoriented fiber sheet-shaped polymer scaffold.
Wherein the concentration of the polyglycolic acid spinning solution is 1-500mg/ml, the concentration of the polyethylene glycol spinning solution is 1-500mg/ml, and the film thickness ratio of the inner layer polyglycolic acid to the outer layer polyethylene glycol is 1:1-5:1. The diameter of the electrostatic spinning fiber is 5nm-50um; the organic solvent may include, but is not limited to, one or more of hexafluoroisopropanol, chloroform, and the like.
(2) Tubular engineering tissue in vitro culture
Planting seed cells in a composite tubular bracket and/or a composite film, and simulating the arterial pulsating pressure and the axial cyclic stretching tension of an organism under a first preset culture condition to perform perfusion culture to obtain tubular and/or sheet engineering tissue consisting of the seed cells and extracellular matrix, wherein the axial cyclic stretching tension condition in the first preset culture condition comprises: firstly, static culturing for 1-10 days, then applying cyclic stretching tension in the axial direction to culture for 6-10 days under the condition of 1-3% of axial deformation, then raising the axial stretching deformation to 5% -7% to continue culturing, and raising the axial stretching deformation to 9% -10% again in the last week of the culturing process.
More specifically, the seed cells are planted in a tubular polymer composite bracket, static culture is carried out for 1-10 days, then arterial pulsating flow pressure perfusion culture is simulated, axial cyclic stretching tension is added, the culture is continued for 4-12 weeks under pulsating flow and cyclic pulsating tension, the extracellular matrix secreted by the seed cells gradually enhances the mechanical property of the pipe wall in the growth and proliferation process of the seed cells on the polymer bracket, meanwhile, the pipe wall polymer is gradually degraded, and finally, the tubular engineering tissue consisting of the seed cells and the extracellular matrix is obtained.
More specifically, the culture conditions for simulating the arterial pulsating flow pressure of the organism are as follows: firstly, culturing in vitro for 1-10 days, then culturing under 10-90mmHg arterial pulse flow for 6-10 days, simultaneously raising arterial pulse flow to 90-130mmHg, and culturing continuously, and raising arterial pulse flow pressure to 130-180mmHg in the last week of culturing process.
Preferably, the in vitro culture period is 4 weeks to 12 weeks, preferably 6 to 12 weeks.
It should be noted that, the addition of the axial cyclic stretching tension and the addition of the arterial pulsating flow pressure of the body are added simultaneously, and the steps are changed simultaneously, and the two are not added sequentially.
The seed cells may include, but are not limited to, one or more of adult human fibroblasts, valve mesenchymal cells and human mesenchymal stem cells including human adipose stem cells, human fibroblasts differentiated from human induced multifunctional stem cells, valve mesenchymal cells, other adult vascular smooth muscle cells of mammalian origin (including pig, cow, sheep, etc.), fibroblasts, valve mesenchymal cells, and mesenchymal stem cells including adipose stem cells, fibroblasts differentiated from induced multifunctional stem cells, valve mesenchymal cells.
More specifically, the cells are derived from a single or multiple donors, or cell banks, using primary-10 generation cells, most preferably primary-6 generation cells, and cultured in vitro for 4 weeks to 12 weeks, most preferably 6 to 10 weeks.
(3) Decellularization treatment
And removing the cells of the tubular and/or sheet engineering tissue to remove the cells of the tube wall, thereby obtaining the tubular engineering tissue and/or sheet engineering membrane composed of extracellular matrix.
The tubular tissue engineering membrane after decellularization mainly consists of extracellular matrix, and extracellular matrix proteins include, but are not limited to, collagen I, collagen III, elastin, and may have trace amounts of polyglycolic acid and/or polyethylene glycol remained.
The decellularization technique may employ chemical reagent treatment. The chemical reagents may be, but are not limited to, 3- [ (3-cholesteryl-propyl) -diethylamine ] -propane sulfonic acid, EDTA-2Na, naCl, naOH and sterile deionized water in the molar concentrations of 1-40mmol/L, 0.5-50mmol/L, 0.01-1.0 mmol/L and 0.1-10.0 mol/L, respectively.
The tubular and/or sheet engineering tissue is subjected to decellularization treatment to obtain a loose 3D extracellular matrix structure, so that the tissue engineering valve is implanted into a body to promote the re-cytozation, the valve formed by the extracellular matrix is ensured to have excellent mechanical properties, and the impact force received during each opening can be borne.
In specific implementation, the tubular and/or sheet engineering tissue is placed into a decellularizing reagent, the treatment is carried out for 1-5 hours at room temperature, then the decellularizing reagent is replaced every 1-5 hours for 2-8 times, preferably 3 times, and then the tubular decellularized matrix tissue engineering membrane is obtained by fully washing with sterile PBS solution.
(4) Cutting out
Cutting the tubular engineering tissue and/or the sheet engineering membrane to obtain an integrated acellular matrix tissue engineering heart valve material or a semi-integrated acellular matrix tissue engineering heart valve material, and combining the integrated acellular matrix tissue engineering heart valve material or the semi-integrated acellular matrix tissue engineering heart valve material with a bracket to form the acellular matrix tissue engineering heart valve prosthesis.
One end of the tubular decellularized matrix tissue engineering membrane is cut into three petal structures 2, namely, a decellularized matrix tissue engineering heart valve 2, as shown in fig. 1, so as to be combined with a stent to make a decellularized matrix tissue engineering heart valve prosthesis.
Example 2 human allogenic decellularized matrix tissue engineering heart valve
The heart valve is composed of extracellular matrix components secreted by seed cells, wherein the extracellular matrix components have in-situ in-vivo re-cytotoxicity.
More specifically, the extracellular matrix protein of the human allogeneic decellularized matrix tissue-engineered heart valve can be, but is not limited to, the first two or three of collagen I, collagen III, or elastin.
The human allogenic decellularized matrix tissue engineering heart valve can be prepared by the preparation method of the human allogenic decellularized matrix tissue engineering heart valve, but is not limited to the method.
Referring to fig. 2 to 4, fig. 4 illustrates a method for manufacturing a surgical valve, specifically, the human allogenic decellularized matrix tissue engineering heart valve includes a decellularized matrix tissue engineering heart valve 2 and a stent 3, and the decellularized matrix tissue engineering heart valve material 2 is sutured or adhered to the stent 3.
Wherein the support 3 is similar to a crown structure, the inner side of each corner of the support 3 is provided with a groove 201, 202 and 203 which can fix the acellular matrix tissue engineering valve, the folding edges 101, 102 and 103 of the acellular matrix tissue engineering heart valve 2 are respectively sewed on the grooves 201, 202 and 203 on the support 3, the three petal arc edges of the acellular matrix tissue engineering valve 2 are just attached to the three arc edges 301, 302 and 303 of the support 3 and sewed and fixed, and the acellular matrix tissue engineering heart valve prosthesis 4 shown in fig. 3 can be obtained, and the acellular matrix tissue engineering heart valve prosthesis 4 in the form is implanted into a human body in a surgical operation mode to replace a diseased mitral valve, tricuspid valve, aortic valve or pulmonary valve.
Example 3 application of human allogeneic decellularized matrix tissue engineering heart valve
The human allogenic acellular matrix tissue engineering heart valve adopts the human allogenic acellular matrix tissue engineering heart valve, and the application of the human allogenic acellular matrix tissue engineering heart valve comprises the embodiment of the human allogenic acellular matrix tissue engineering heart valve, and the beneficial effects of the human allogenic acellular matrix tissue engineering heart valve can be applied to the application of the human allogenic acellular matrix tissue engineering heart valve. The human allogenic decellularized matrix tissue engineering heart valve is combined with a valve stent and then applied to a conventional surgical heart valve replacement operation or an interventional heart valve replacement operation of a mitral valve, a tricuspid valve, an aortic valve and a pulmonary valve. Referring to fig. 7, 71 in fig. 7 is an acellular matrix tissue engineering heart valve, 72a and 72b are valve endothelial cells, and 73 is valve interstitial cells, and it can be seen from fig. 7 that the tissue engineering heart valve can realize rapid endothelialization of the surface of the valve leaflet, re-cellularization of the valve interstitial cells into the internal tissue of the valve leaflet after being implanted into a body, and in-situ reconstruction of the new heart valve in the body.
The human allogenic decellularized matrix tissue engineering heart valve is combined with a valve stent and then applied to surgical replacement or interventional replacement.
Referring to fig. 5 and 6, fig. 5 illustrates a method for manufacturing an interventional valve, specifically, a human allogenic decellularized matrix tissue engineering heart valve includes a decellularized matrix tissue engineering heart valve 2 and a valve stent 5, and the decellularized matrix tissue engineering heart valve material 2 is sutured or bonded to the valve 5.
Wherein the valve support 5 is a reducing reticular tube, the decellularized matrix tissue engineering heart valve 2 is arranged in the valve support 5, the folding edges 101, 102 and 103 of the valve are fixed on the support with the smaller diameter at one end in a sewing mode, and the three petal arc edges of the valve are fixed on the support with the larger diameter at one end in the same sewing mode, so that the decellularized matrix tissue engineering heart valve prosthesis 6 shown in fig. 5 can be obtained. This form of decellularized matrix tissue engineering heart valve prosthesis 6 requires replacement of a diseased mitral valve, tricuspid valve, aortic valve, or pulmonary valve by implantation into the body via a catheter-based approach that may employ self-expanding and balloon-expanding approaches.
It will be apparent that the embodiments described above are merely some, but not all, embodiments of the invention. Based on the embodiments of the present invention, those skilled in the art may make other different changes or modifications without making any creative effort, which shall fall within the protection scope of the present invention.
Claims (10)
1. A method for preparing a heart valve of human allogeneic decellularized matrix tissue engineering, comprising the steps of:
preparing a composite tubular bracket and/or a composite film of polyglycolic acid and polyethylene glycol polymer by adopting electrostatic spinning;
planting seed cells in a composite tubular bracket and/or a composite film, and simulating the arterial pulsating pressure and the axial cyclic stretching tension of an organism under a first preset culture condition to perform perfusion culture to obtain tubular and/or sheet engineering tissue consisting of the seed cells and extracellular matrix, wherein the axial cyclic stretching tension condition in the first preset culture condition comprises: firstly, statically culturing for 1-10 days, then applying cyclic stretching tension in the axial direction to culture for 6-10 days under the condition of 1-3% of axial deformation, then raising the axial stretching deformation to 5% -7% to continue culturing, and raising the axial stretching deformation to 9% -10% again in the last week of the culturing process;
removing cells from the tubular and/or sheet-like engineering tissue to remove cells from the tubular wall, thereby obtaining a tubular-like engineering tissue and/or sheet-like engineering membrane composed of extracellular matrix;
cutting the tubular engineering tissue and/or the sheet engineering membrane to obtain a valve leaflet material of the acellular matrix tissue engineering heart valve, and preparing the acellular matrix tissue engineering heart valve by using the valve leaflet material, wherein the acellular matrix tissue engineering heart valve comprises a conventional surgical heart valve and a minimally invasive intervention heart valve.
2. The method for preparing the heart valve of the human allogeneic decellularized matrix tissue engineering according to claim 1, wherein the step of planting the seed cells in the composite tubular stent and/or the composite film and performing perfusion culture under the condition of simulating the pulsating flow pressure of the body artery and the axial cyclic stretching tension under the first preset culture condition to obtain the tubular and/or sheet engineering tissue consisting of the seed cells and the extracellular matrix comprises the following steps:
the seed cells are planted in the composite tubular bracket and/or the composite film, and the tubular and/or sheet engineering tissue consisting of the seed cells and extracellular matrix is obtained by simulating the arterial pulsating flow pressure and the axial cyclic stretching tension of the organism under the first preset culture condition and carrying out perfusion culture for 4-12 weeks.
3. The method for preparing the heart valve of the human allogeneic decellularized matrix tissue engineering according to claim 2, wherein the step of planting the seed cells in the composite tubular stent and/or the composite film and performing perfusion culture under the condition of simulating the arterial pulsating flow pressure and the axial cyclic stretching tension of the body under the first preset culture condition to obtain the tubular and/or sheet engineering tissue composed of the seed cells and the extracellular matrix, the first preset culture condition simulating the arterial pulsating flow pressure culture condition of the body comprises:
firstly, static culturing for 1-10 days, then culturing for 6-10 days under arterial pulse flow with pressure of 0-90mmHg, then raising arterial pulse flow to 90-130mmHg, continuously culturing, and raising arterial pulse flow pressure to 130-180mmHg in the last week of the culturing process.
4. The method for preparing the heart valve of the human allogeneic decellularized matrix tissue engineering according to claim 1, wherein the inner layer of the composite tubular stent and/or the composite film is polyglycolic acid, the outer layer is polyglycolic acid polymer, and the thickness ratio of the polyglycolic acid of the inner layer to the polyglycolic acid polymer of the outer layer is 1:1-5:1.
5. The method for preparing the heart valve of the human allogeneic decellularized matrix tissue engineering according to claim 1, wherein the step of preparing the composite tubular stent and/or the composite film of polyglycolic acid and polyethylene glycol polymer by adopting electrostatic spinning specifically comprises the following steps:
preparing a composite tubular bracket of polyglycolic acid and polyethylene glycol polymer by adopting electrostatic spinning, wherein the inner diameter of the composite tubular bracket is 18-30mm, the total thickness of the pipe wall is 0.2-2mm, the length is 2-10cm, the porosity of the pipe wall of the bracket is 80-95%, and the aperture is 50nm-200um;
or the steps for preparing the composite tubular stent and/or the composite film of polyglycolic acid and polyethylene glycol polymer by adopting electrostatic spinning are specifically as follows:
and preparing a composite film of polyglycolic acid and polyethylene glycol polymer by adopting electrostatic spinning, wherein the total thickness of the composite film is 0.5-2mm, the length is 2-10cm, the width is 6-9 cm, the porosity of the bracket is 80-95%, and the aperture is 50nm-200 mu m.
6. The method for preparing a heart valve of the human allogeneic decellularized matrix tissue engineering according to claim 1, wherein the step of the decellularizing method in the step of removing tubular wall cells from the tubular and/or sheet-shaped engineering tissue to obtain the tubular-shaped engineering tissue and/or sheet-shaped engineering membrane composed of extracellular matrix specifically comprises the following steps:
placing the tubular and/or sheet-shaped engineering tissue into a configured decellularized reagent, and treating for 1-3h at room temperature; changing the decellularized reagent once every 1-3 hours for 3-6 times to obtain tubular engineering tissue and/or sheet engineering membrane composed of extracellular matrix; the decellularized reagent is prepared from 3- [ (3-cholesteryl amidopropyl) -diethylamine ] -propane sulfonic acid, disodium ethylenediamine tetraacetate (EDTA-2 Na), naCl, naOH and sterile deionized water.
7. The method of claim 1, wherein the seed cells comprise one or more of fibroblasts from adult humans or mammals, valve stromal cells, mesenchymal stem cells and fibroblasts derived from induction of differentiation of multifunctional stem cells, and valve stromal cells.
8. The method of claim 1, wherein the seed cells are derived from a single or multiple donors or cell banks using primary-10 generation cells.
9. A human allogeneic decellularized matrix tissue engineering heart valve prepared by the method of preparing a human allogeneic decellularized matrix tissue engineering heart valve according to claims 1-8.
10. The application of the human allogeneic decellularized matrix tissue engineering heart valve is characterized in that the human allogeneic decellularized matrix tissue engineering heart valve is combined with a valve support and then applied to a conventional surgical heart valve replacement or interventional heart valve replacement of mitral valve, tricuspid valve, aortic valve and pulmonary valve, and the human allogeneic decellularized matrix tissue engineering heart valve can realize rapid endothelialization of the surface of a valve leaflet and decellularization of the internal tissue of the valve leaflet after being implanted into a body due to the characteristic structure of the human allogeneic decellularized matrix tissue engineering heart valve, and the new heart valve is reconstructed in situ in the body.
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