CN116159185B - Composite acellular matrix material and preparation method thereof - Google Patents
Composite acellular matrix material and preparation method thereof Download PDFInfo
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- CN116159185B CN116159185B CN202211678118.0A CN202211678118A CN116159185B CN 116159185 B CN116159185 B CN 116159185B CN 202211678118 A CN202211678118 A CN 202211678118A CN 116159185 B CN116159185 B CN 116159185B
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- 239000002131 composite material Substances 0.000 title claims abstract description 91
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
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- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- 241001465754 Metazoa Species 0.000 claims description 19
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/60—Materials for use in artificial skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- 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
- A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3633—Extracellular matrix [ECM]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Dermatology (AREA)
- Transplantation (AREA)
- Epidemiology (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Botany (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Urology & Nephrology (AREA)
- General Chemical & Material Sciences (AREA)
- Prostheses (AREA)
Abstract
The invention provides a composite acellular matrix material capable of enhancing mechanical properties and a preparation method thereof. The composite acellular matrix material is formed by compositing two or more layers of acellular matrixes. Taking a two-layer composite acellular matrix as an example: the composite acellular matrix material comprises a first acellular matrix layer and a second acellular matrix layer, wherein a first embedded part is formed on the surface of the first acellular matrix layer facing the second acellular matrix layer, a second embedded part is formed on the surface of the second acellular matrix layer facing the first acellular matrix layer, the first embedded part is embedded with the second embedded part, one of the first embedded part and the second embedded part comprises a protrusion, and the other one of the first embedded part and the second embedded part comprises a groove.
Description
Technical Field
The invention relates to a composite acellular matrix material and a preparation method thereof.
Background
Extracellular matrix (ECM) refers to a substance that is located around the lower layers of epithelial or endothelial cells, connective tissue cells, and provides mechanical support and physical strength for the integrity of tissues, organs, and even the whole body. Acellular matrix refers to the fibrous network of extracellular matrix of human or animal tissue of different origin, from which the relatively weakly immunogenic components of the tissue have been removed by a decellularization process. The skeleton of extracellular matrix mainly comprises proteins, and the core component is collagen, and contains proteoglycan, glycoprotein, growth factor, etc. It not only provides mechanical support and physical strength for the body, but also has great influence on cell adhesion, growth and differentiation.
The acellular matrix has extremely low immunogen and natural tissue support structure, and can be gradually replaced by the tissue of a patient along with the extension of time after being implanted into a human body, so that the tissue regeneration of the organism is guided. From a source, currently decellularized matrix products are mainly derived from allogeneic (human) tissue or xenogeneic tissue. However, due to the limited source of the foreign tissue, the wide range of applications of the decellularized foreign tissue is limited. The heterogeneous decellularized tissue is concerned by more and more scholars and medical field manufacturers at home and abroad due to the characteristics of rich sources, low immunogenicity and the like. Animal tissues such as decellularized pigskin, cow leather, pig intestinal submucosa mucosa, cow pericardium film, fish skin and the like have been developed into medical products and successfully applied to clinic, and have extremely high potential in the aspects of difficult healing wound surface repair, residual cavity filling, soft tissue mechanics reinforcement and the like.
The mechanical property of a large short plate of the acellular matrix material is insufficient, which is particularly obvious in soft tissue reinforced products. Such as pelvic floor patches, the acellular matrix is always not broken through in this field of application due to the high recurrence rate. This is related to the relatively thin acellular matrix and the insufficient mechanical properties. Such as fish skin, the application of the acellular dermal matrix of fish is often limited due to the generally low thickness of the fresh water or sea water fish skin. According to the method disclosed in the Chinese patent publication No. CN112618799A, the fishskin is pretreated, and then subjected to washing, disinfection, decellularization treatment, virus inactivation treatment, heavy metal removal treatment, protection solution treatment, sterilization and freeze-drying to obtain the fishskin decellularized dermis matrix. The tilapia skin and salmon skin mentioned in the examples and comparative examples of the patent are very thin, so that the upper limit of mechanical properties exists in practical application of the fish skin materials, and therefore, a certain short plate exists in applications with requirements on mechanics such as soft tissue reinforcement.
At present, the mechanical reinforcement and thickening mode of the acellular matrix product is usually lamination, namely, a plurality of matrixes are overlapped through physical adsorption, and finally, the matrixes are freeze-dried and fixed, so that the acellular matrix product is more commonly applied to film-shaped matrix products. For example, the composite multi-layer acellular matrix can be obtained by laminating multiple layers of mucous membranes of the lower layers of the small intestines of pigs, pressing and drying. However, such matrix films that can be simply laminated are generally thinner, even if laminated to a much smaller thickness than non-film matrices, such as animal dermis. The thickness of the acellular matrix is more favorable for migration, growth and differentiation of cells and is generally more advantageous in terms of mechanical properties. However, many decellularized substrates, such as dermal substrates, generally have a relatively smooth surface after treatment, and relatively weak adhesion and cell adhesion. In addition, due to the water swelling property of the bracket structure, the two acellular matrixes are not easy to keep in a laminated state, and are easy to slip after being attached, so that the mechanical properties of the two acellular matrixes are difficult to develop.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a composite acellular matrix material capable of enhancing mechanical properties and a preparation method thereof.
The first aspect of the present invention provides a composite acellular matrix material comprising a first acellular matrix layer and a second acellular matrix layer, wherein a first fitting portion is formed on a surface of the first acellular matrix layer facing the second acellular matrix layer, a second fitting portion is formed on a surface of the second acellular matrix layer facing the first acellular matrix layer, the first fitting portion and the second fitting portion are fitted, one of the first fitting portion and the second fitting portion comprises a protrusion, and the other one of the first fitting portion and the second fitting portion comprises a groove.
By adopting the composite acellular matrix material, the first acellular matrix layer and the second acellular matrix layer are firmly combined together under the embedding action of the first embedding part and the second embedding part, for example, the first acellular matrix layer and the second acellular matrix layer are not easy to separate even after long-time soaking, so that the composite acellular matrix material has multiple mechanical properties compared with a single acellular matrix and has stable performance.
In addition, the composite acellular matrix material has the technical effect of better safety, and is specifically as follows:
as other structures by compounding the acellular matrix material, it is conceivable to fix a plurality of pieces of acellular matrix by multi-point suturing, so as to achieve the purpose of thickening the laminate and maintaining mechanical properties. The suture used in the mode introduces new materials, so that the suture of the product is increased, and the potential safety hazard is increased. Further, the addition of stitches necessarily increases the complexity of the manufacturing process.
In addition, it is also considered to use a biological glue, such as collagen, gelatin, or other adhesive biological glue, to adhere two pieces of acellular matrix, so as to achieve the effects of thickening and maintaining mechanical properties. This method is theoretically viable, but has poor practical adhesion, is not stable in liquid for long periods of time in a partially decellularized matrix, and provides limited mechanical properties (only slightly higher than a monolithic decellularized matrix). In addition, the method introduces new substances of the biological glue, so that the problems of glue brushing uniformity, glue brushing thickness influence and the like are also caused, and in addition, the new substances can also increase potential safety hazards of products.
By adopting the composite acellular matrix material, the mechanical property is maintained through the embedded structure of the first acellular matrix layer and the second acellular matrix layer, and on the basis of obtaining good mechanical property, a new material is not added, so that the safety is good.
As one possible implementation manner of the present invention, the first engaging portion and the second engaging portion are square, circular, trapezoidal, triangular or other irregular patterns in cross section.
As one possible implementation manner of the present invention, there are a plurality of the first fitting portions and the second fitting portions, and the plurality of the first fitting portions and the plurality of the second fitting portions are the same or different in size in the layer thickness direction and/or in a direction perpendicular to the layer thickness direction.
As one possible implementation manner of the present invention, the first engaging portion and the second engaging portion are plural, and the shapes of the plural first engaging portions and the plural second engaging portions are the same or different.
As one possible implementation manner of the present invention, the first engaging portion and the second engaging portion are provided in plural, and the first engaging portion and the second engaging portion are each arranged in a wavy, zigzag or square waveform as viewed in cross section.
As one possible implementation manner of the present invention, the composite acellular matrix material further includes a third acellular matrix layer, the second acellular matrix layer is disposed between the third acellular matrix layer and the first acellular matrix layer, a third engaging portion is formed on a surface of the third acellular matrix layer facing the second acellular matrix layer, a fourth engaging portion is formed on a surface of the second acellular matrix layer facing the third acellular matrix layer, the third engaging portion and the fourth engaging portion are engaged, one of them includes a protrusion, and the other includes a groove.
As one possible implementation manner of the present invention, the shapes of the second fitting portion and the fourth fitting portion are the same or different.
As one possible implementation of the present invention, the first decellularized matrix layer and/or the second first decellularized matrix layer is derived from a human or an animal including a terrestrial mammal including, but not limited to, pigs, cattle, sheep or horses, or an aquatic animal including, but not limited to, teleosts, cartilages or aquatic mollusks.
The second aspect of the present invention provides a method for preparing a composite acellular matrix material, comprising the steps of: a fitting portion forming step of forming a first fitting portion on a surface of a first acellular matrix and forming a second fitting portion on a surface of a second acellular matrix, wherein one of the first fitting portion and the second fitting portion includes a projection and the other includes a recess; and a lamination step of bonding the first acellular matrix and the second acellular matrix in a state in which the first fitting portion and the second fitting portion are fitted to each other, thereby obtaining a laminate, and obtaining a composite acellular matrix material from the laminate.
In the preparation method of the present invention, a first fitting portion is formed on the surface of a first acellular matrix, a second fitting portion is formed on the surface of a second acellular matrix, and then the first acellular matrix and the second acellular matrix are bonded to each other in a state in which the first fitting portion and the second fitting portion are fitted to each other, whereby a laminate is obtained, and a composite acellular matrix material is obtained from the laminate. Therefore, in the obtained composite acellular matrix material, the layer formed by the first acellular matrix and the layer formed by the first acellular matrix can be strongly bonded together under the embedding action of the first embedding part and the second embedding part, so that the mechanical property of the composite acellular matrix material can be maintained.
In addition, the technical effects substantially the same as those of the first aspect can be obtained by the production method of the second aspect, and thus will not be further explained here.
As one possible implementation of the present invention, in the fitting portion forming step, the first fitting portion and/or the second fitting portion is formed by embossing with a template or etching with a laser.
As a possible implementation of the present invention, the template has a lattice-like or wave-like concave-convex pattern.
As one possible implementation of the present invention, the first fitting portion and the second fitting portion are square, circular, triangular or trapezoidal in cross section.
As one possible implementation of the present invention, before the fitting portion forming step, the method further includes: and a surface treatment step of roughening the surface of the first acellular matrix and/or the surface of the second acellular matrix.
Thus, the roughness of the surface of the acellular matrix is improved, the surface area contacted with the wound surface is increased, and the adhesiveness of the material and the mobility of cells in the material are improved.
As a possible implementation of the present invention, the surface roughening treatment is performed by applying a chemical agent or by physical shaping.
As one possible implementation of the present invention, the chemical agent includes an acid or an oxidizing agent, wherein the acid includes but is not limited to acetic acid, hydrochloric acid or fruit acid, and the oxidizing agent includes but is not limited to hydrogen peroxide or peracetic acid.
As one possible implementation of the present invention, the physical shaping means includes laser processing, tool etching, or rough plate polishing.
It will be appreciated that after the laminate is obtained, it may be subjected to further processing to obtain a composite decellularized matrix material, some examples of which are given below.
As a possible implementation manner of the present invention, after the lamination process, the method further includes: and a fixing step of crosslinking the laminate.
Thus, the first fitting portion and the second fitting portion can maintain a good fitting action.
Specifically, since the decellularized base is a stent material, the water absorption volume is expanded, and thus, even a texture (concave-convex structure) formed by embossing or the like may be restored after a period of immersion. In contrast, in the invention, the bonding connection of the internal structure of the acellular matrix is increased through crosslinking, and after the reaction, the surface of the acellular matrix is not easy to restore the original flat structure, so that the first embedded part and the second embedded part maintain good embedded effect, and the composite acellular matrix material maintains good mechanical property.
The fixing step is preferably performed under dehydration.
As one possible implementation of the present invention, in the fixing step, crosslinking is performed in a state where the laminate is sandwiched by sandwiching plates.
Thus, the engaging portion of the acellular matrix is not easily deformed by restoration, and the fixation effect can be improved.
As one possible implementation of the invention, the laminate is crosslinked using one or more of formaldehyde, glutaraldehyde, epoxide, genipin, carbodiimide, hydroxysuccinimide, hexamethylene diisocyanate, acyl azide and solutions of diphenyl phosphate, glutamine transaminase.
As a possible implementation manner of the present invention, after the lamination process, the method further includes: and an expansion step of immersing the laminate in an expansion liquid.
Therefore, the first jogged part and the second jogged part can absorb water to increase the volume and improve the friction between the two parts, so that the good jogged effect can be maintained, and the composite acellular matrix material can maintain good mechanical properties.
The expansion step is preferably performed after the fixing step.
As one possible implementation of the present invention, the swelling solution includes, but is not limited to, purified water, phosphate buffer, borate buffer, or tris hydrochloride buffer.
As one possible implementation of the present invention, before the fitting portion forming step, the method further includes: an outer matrix tissue collection step of collecting an outer matrix tissue of an animal from the animal, and washing the outer matrix tissue to remove impurities other than the matrix; and a decellularized matrix preparation step of performing decellularized treatment on the outer matrix tissue to obtain the first decellularized matrix and the second decellularized matrix.
As a possible implementation manner of the present invention, the cleaning in the external matrix tissue collection procedure includes removing fat, pigment, inorganic salt or attachment with a cleaning agent.
As one possible implementation of the present invention, the cleaning agent includes one or more of an alcohol, an alkali solution, an acid solution, a salt solution, an oxidizing agent, or an adsorbent, the alcohol includes ethanol or isopropanol, the alkali solution includes sodium hydroxide or potassium hydroxide solution, the acid solution includes acetic acid or citric acid, the salt solution includes sodium chloride or potassium chloride, the oxidizing agent includes hydrogen peroxide or potassium permanganate, and the adsorbent includes activated carbon or macroporous resin.
As a possible implementation manner of the present invention, in the outer matrix tissue collecting procedure, the outer matrix tissue is cut to form square, round, rectangular, trapezoidal hollow circular rings or other irregular patterns.
As a possible implementation manner of the present invention, in the decellularized matrix preparation process, the outer matrix tissue is decellularized by a physical method or a chemical reagent method, wherein the physical method comprises ultrasonic wave, freeze thawing, mechanical friction, mechanical stirring or pressurizing, and the chemical reagent method uses reagents including salt solution, acid, alkali, oxidant, surfactant and an agent capable of destroying cell membranes or binding DNA in enzyme.
As a possible implementation of the present invention, the first decellularized matrix and/or the second first decellularized matrix is derived from a terrestrial mammal or an aquatic animal.
The composite acellular matrix material of the invention includes, but is not limited to, allogeneic (human) or xenogeneic acellular dermal matrix, acellular visceral membranes, acellular bone matrix and the like, and xenogeneic acellular dermal matrix is preferred. The xenogeneic decellularized matrix source includes, but is not limited to, terrestrial mammals (e.g., pigs, cattle, sheep, horses, etc.), aquatic animals (e.g., teleosts, cartilaginous fish, aquatic mollusks, etc.), and preferably has a thickness of animal tissue.
In the prior art, for example, 2 ECMs simply stacked are generally only one piece of ECMs to function as detected mechanics. For example, one ECM break strength is 100N, and two sheets simply stack up to 100-120N, but not 200N. Compared with the prior art, the invention can provide the composite acellular matrix material which is thicker than the original matrix material, better in mechanical property and stronger in adhesiveness. The material overcomes the defects of part of matrix materials such as smooth surface and poor laminating property, and improves cell adhesion by increasing surface roughness; the embedded structure which can firmly attach a plurality of acellular matrixes is manufactured, so that the mechanical properties of the composite acellular matrix material can be maintained. In addition, the multiple acellular matrixes are fully combined by chemical fixation, physical expansion and other methods, so that the effects of thickening and mechanical reinforcement of the product are achieved. Different from simply superposing two acellular matrixes, the composite acellular matrix material can keep an integrated structure in liquid for a long time, and the superposed matrixes are not easy to separate. In mechanical aspect, the composite acellular matrix material can provide more uniform mechanical strength than a simple stacked matrix, the mechanical improvement effect of the material is not poor due to uneven stress, and the effect of 1+1=2 can be achieved.
In addition, the acellular matrix improves the roughness of the surface of the acellular matrix, increases the surface area contacted with a wound surface and improves the adhesiveness of materials and the mobility of cells in the materials through a special surface treatment technology; the surface area of contact between two acellular matrixes is increased by the treatment modes of surface stamping or physical etching, chemical fixation and physical expansion, so that the two acellular matrixes can be closely attached, and the thickness and mechanical properties of the combined product are obviously increased. The composite acellular matrix material provided by the invention improves the short plates with insufficient mechanical properties of the acellular matrix material to a certain extent, so that the composite acellular matrix material has more application value in the fields of soft tissue reinforcement, wound repair and the like.
Drawings
The individual technical features of the present application and their relationships are further described below with reference to the accompanying drawings. The drawings are exemplary, some technical features are not shown in actual proportion, and some drawings may omit technical features that are conventional in the art to which the present application pertains and are not essential to understanding and realizing the present application, or additionally show technical features that are not essential to understanding and realizing the present application, that is, combinations of the technical features shown in the drawings are not limiting the present application. In addition, throughout this application, like reference numerals refer to like elements. The specific drawings are as follows:
FIG. 1 is a schematic illustration of a cross-sectional structure of a composite decellularized matrix material of an embodiment of the invention;
FIG. 2 is a schematic representation of a cross-sectional structure of a composite decellularized matrix material of an embodiment of the invention;
FIG. 3 is a schematic representation of the cross-sectional structure of a composite decellularized matrix material of an embodiment of the invention;
FIG. 4A is a schematic diagram of an exploded structure of a composite decellularized matrix material of an embodiment of the invention;
FIG. 4B is a plan view of the lower acellular matrix layer of FIG. 4A;
FIG. 5A is a schematic diagram of an exploded structure of a composite decellularized matrix material of an embodiment of the invention;
FIG. 5B is a schematic illustration of a cross-sectional structure of the composite decellularized matrix material of FIG. 5A;
FIG. 6 is a schematic representation of a cross-sectional structure of a composite decellularized matrix material of an embodiment of the invention;
FIG. 7 is a schematic representation of the cross-sectional structure of a composite decellularized matrix material of an embodiment of the invention;
FIG. 8 is a schematic representation of the cross-sectional structure of a composite decellularized matrix material of an embodiment of the invention;
FIG. 9 is a schematic representation of a cross-sectional structure of a composite decellularized matrix material of an embodiment of the invention;
FIG. 10 is a graph showing the results of suture tension experiments of example 1, comparative example 1.1, and comparative example 1.2;
FIG. 11 is the experimental data showing the effect of the surface treatment according to example 4 of the present invention on cell adhesion, which shows the adhesion proliferation of mouse fibroblasts on decellularized matrix.
Detailed Description
Next, a specific embodiment of the present invention will be described in detail.
The embodiment provides a preparation method of a composite acellular matrix material, which comprises the following steps:
(1) An outer matrix tissue collection procedure: collecting animal outer matrix tissue on animal, cleaning to remove non-matrix impurities, and cutting into regular shape.
(2) Acellular matrix preparation procedure: and (3) carrying out decellularization treatment on the animal outer matrix tissue to obtain the decellularized matrix.
(3) Surface treatment process: one or both surfaces of the acellular matrix are subjected to surface roughening treatment, and the acellular matrix may be washed and dehydrated after the surface roughening treatment.
(4) Fitting portion forming step: the plurality of acellular substrates are molded by means of template stamping, laser etching, or the like, and a concave-convex structure (fitting portion) is formed on the surface of each acellular substrate.
In this case, two acellular substrates are exemplified, and one of them may be formed with a protrusion and the other may be formed with a groove, or both may be formed with a protrusion and a groove (for example, a wave-shaped structure or the like described later). Wherein the depth of the groove accounts for 40% -50% of the thickness of the acellular matrix.
(5) Overlapping procedure: the acellular matrix was aligned and fitted in pairs, and the acellular matrix was bonded to each other in pairs in this state, to obtain a laminate.
(6) Fixing procedure: the laminate may be fixed (crosslinked) with a chemical agent for a while in a state of being sandwiched by the sandwiching plates.
(7) Expansion process: then, soaking the laminated body in an expansion liquid to remove cell matrixes for a period of time; wherein the state of being held by the clip can be continued.
(8) Post-treatment procedure: and (3) cleaning and freeze-drying the stack body of the acellular matrix, cutting the stack body into a certain regular shape, and packaging and sterilizing by irradiation to obtain a finished product, namely the composite acellular matrix material.
By adopting the preparation method, as the concave-convex structure (the embedded part) is formed on the surface of the acellular matrix, after lamination, the acellular matrixes can be firmly combined together under the embedded action of the concave-convex structure, and the problems of separation, stripping and the like are not easy to occur, so that the thickened composite acellular matrix material can be obtained, and meanwhile, the mechanical property of the composite acellular matrix material can be maintained.
In addition, in this embodiment, the surface roughening treatment improves the surface roughness of the acellular matrix, increases the surface area in contact with the wound bed, and improves the adhesiveness of the material and the mobility of cells in the material.
In more detail, in the case of decellularized matrix products, the cell adhesion and the thickness of the product often determine the performance of the product in terms of tissue repair promotion and mechanical reinforcement. After some acellular matrix (such as acellular dermal matrix) is treated, the dermis layer is smooth on both sides, the surface area is relatively low, the material adhesion is poor, and the adhesiveness of cells on the material may be weak. As in chinese patent publication CN113827776a, after cutting the dermis layer of animal raw skin, degreasing with nonionic surfactant, removing cells with alkali solution, balancing the pH of the material with buffer solution, and finally harvesting the decellularized dermis matrix. The acellular dermal matrix prepared by the method has smooth two surfaces, low surface area contacted with the wound coverage surface and relatively poor cell adhesion. For this reason, in the present embodiment, the surface area of the decellularized matrix in contact with the wound surface is increased by the surface roughening treatment, and cell adhesion can be enhanced.
In the present embodiment, the laminate is subjected to the immobilization treatment (i.e., the immobilization step), so that the firmness of the fitting structure can be improved and a satisfactory fitting effect can be maintained.
Specifically, since the decellularized base is a stent material, the water absorption volume is expanded, and thus, even a texture (concave-convex structure) formed by embossing or the like may be restored after a period of immersion. In contrast, in the invention, the bonding connection of the internal structure of the acellular matrix is increased by crosslinking, and after the reaction, the surface of the acellular matrix is not easy to recover the original flat structure, so that the embedded concave-convex structure (embedded part) maintains good embedding effect, and the composite acellular matrix material maintains good mechanical property.
The fixing step is preferably performed under dehydration.
In addition, the crosslinking is performed in a state of clamping the clamping plate, so that the fixing effect can be improved.
In the present embodiment, the laminate is further subjected to an expansion treatment in the expansion step, and thus the volume of the laminate can be increased by absorbing water in the concave-convex structure (fitting portion), and friction between the fitting portions can be improved, so that a favorable fitting effect can be maintained, and favorable mechanical properties of the composite acellular matrix material can be maintained.
The expansion step is preferably performed after the fixing step.
In addition, it can be understood that this embodiment also provides a composite acellular matrix material, wherein the surface of the acellular matrix layer has a concave-convex structure (a fitting portion), the concave-convex structure (a fitting portion) between layers is fitted, and the acellular matrix layer can be strongly bonded under the fitting effect, so that the problems of separation, peeling and the like are not easy to occur, and the mechanical properties of the composite acellular matrix material are maintained.
Optionally, the washing in the step (1) means removing impurities such as fat, pigment, inorganic salt, and attached matter of the acellular matrix with a chemical reagent (washing reagent).
Optionally, the cleaning agent in the step (1) comprises one or more of alcohols such as ethanol and isopropanol, alkali solutions such as sodium hydroxide and potassium hydroxide, weak acids such as acetic acid and citric acid, salt solutions such as sodium chloride and potassium chloride, oxidants such as hydrogen peroxide and potassium permanganate, adsorbents such as activated carbon and macroporous resin.
Optionally, the certain regular shape in the step (1) includes a regular pattern, such as a square, a circle, a rectangle, a trapezoid, and the like, and also includes an irregular pattern, such as a hollowed-out ring, and the like.
Optionally, the decellularization treatment in step (2) is aimed at removing cells within the matrix, including physical methods such as ultrasound, freeze thawing, mechanical friction, mechanical agitation, pressurization; including chemical methods such as high concentration salt solutions, acids, bases, oxidants, surfactants, enzymes and the like, which disrupt cell membranes or bind DNA.
Optionally, the surface roughening treatment in the step (3) is performed by applying a chemical agent or by physical shaping.
Optionally, the chemical reagent is acid or oxidant such as acetic acid, hydrochloric acid, fruit acid, hydrogen peroxide or peracetic acid, etc., and the dropper is used to uniformly smear the cell-free matrix surface with proper amount of chemical reagent, and react for a period of time.
Optionally, physical shaping comprises laser treatment, cutter etching, rough plate polishing and the like, and the removed matrix is removed by purified water after shaping.
Optionally, the template embossing in the step (4) is to press the surface treated with a mold having a certain rugged pattern, so as to leave rugged marks on the surface of the acellular matrix.
Alternatively, the pattern of the stamp plate may be a dot matrix, a wave, or other patterns that provide good engageability of raised and recessed portions of the decellularized matrix after stamping.
Optionally, the laser etching in the step (4) is to etch the surface of the acellular matrix with laser to form a groove structure, and the groove may be columnar or punctiform, and the cross-sectional shape may be trapezoid, circle, square or other irregular patterns.
Optionally, the fixing of the chemical reagent in the step (6) is fixing the structure of the decellularized matrix with the surface mutually embedded by adopting one or more of formaldehyde, glutaraldehyde, epoxide, genipin, carbodiimide, hydroxysuccinimide, hexamethylene diisocyanate, acyl azide, diphenyl phosphate and glutamine transaminase.
Optionally, the expansion liquid in the step (7) is one of purified water, phosphate buffer, borate buffer and tris hydrochloride buffer.
Compared with the prior art, the specific embodiment of the invention can provide the composite acellular matrix material which is thicker than the original matrix material, has better mechanical property and stronger wound adhesion. The material overcomes the defects of part of matrix materials such as smooth surface and poor laminating property, and improves cell adhesion by increasing surface roughness; the embedded structure which can firmly attach a plurality of acellular matrixes is manufactured, so that the mechanical properties of the composite acellular matrix material can be maintained. In addition, the multiple acellular matrixes are fully attached by chemical fixation, physical expansion and other methods, so that the effects of thickening and mechanical reinforcement of the product are achieved. Different from simply superposing two acellular matrixes, the composite acellular matrix material can keep an integrated structure in liquid for a long time, and the superposed matrixes are not easy to separate. In mechanical aspect, the composite acellular matrix material can provide more uniform mechanical strength than a simple stacked matrix, the mechanical improvement effect of the material is not poor due to uneven stress, and the effect of 1+1=2 can be achieved.
In addition, the acellular matrix of the specific embodiment of the invention improves the roughness of the surface of the acellular matrix, increases the surface area contacted with a wound surface and improves the adhesiveness of materials and the mobility of cells in the materials through a special surface treatment technology; the surface area of contact between two acellular matrixes is increased by the treatment modes of surface stamping or physical etching, chemical fixation and physical expansion, so that the two acellular matrixes can be closely attached, and the thickness and mechanical properties of the combined product are obviously increased. The composite acellular matrix material provided by the invention improves the short plates with insufficient mechanical properties of the acellular matrix material to a certain extent, so that the composite acellular matrix material has more application value in the fields of soft tissue reinforcement, wound repair and the like.
Alternatively, the composite acellular matrix material of the present embodiment has a rough surface on one side and a smooth surface on the other side, and the rough surface may be formed by fibrous filaments or irregular fibrous protrusions.
Alternatively, the composite decellularized matrix material of the present embodiment can thicken by more than 1.5 times compared to a monolayer of cell matrix.
Alternatively, the composite acellular matrix material of the present embodiment is composed of at least two pieces of acellular matrix, and the processing technology of each piece may be the same or different.
Alternatively, the composite decellularized matrix material of this embodiment has a mechanical strength of at least 2 times that of a monolayer.
Optionally, the composite decellularized matrix material of the embodiment is not easily separated after being soaked in water for at least 168 hours.
The concave-convex structure (fitting portion) of the present embodiment may take various forms. Some specific examples are described below with reference to fig. 1-9.
The structure of the chimeric portion of a plurality of different composite decellularized matrix materials is schematically shown in FIGS. 1-6.
FIG. 1 is a schematic diagram of a cross-sectional (cross-section perpendicular to the layer thickness) structure of a composite acellular matrix material according to one embodiment of the invention. In the example of fig. 1, the composite acellular matrix material has an acellular matrix layer 1 and an acellular matrix layer 2. A plurality of identical grooves 11 are formed on the surface of the decellularized matrix layer 1 facing the decellularized matrix layer 2, and protrusions are formed between adjacent grooves 11, and are integrally arranged in a wavy line shape. A plurality of identical protrusions 21 are formed on the surface of the decellularized matrix layer 2 facing the decellularized matrix layer 1, grooves are formed between the protrusions 21, and the entire surface is arranged in a wavy line shape. The grooves 11 and the protrusions 21 are fitted to each other, so that the bonding strength between the acellular matrix layer 1 and the acellular matrix layer 2 can be improved.
In addition, the grooves 11 and the protrusions 21 are mountain-shaped, and the tops thereof are smoothly transited.
FIG. 2 is a schematic representation of the cross-sectional structure of a composite decellularized matrix material of an embodiment of the invention. In the example of fig. 2, the acellular matrix layer 1A has a plurality of projections 11A, and the plurality of projections 11A are mountain-shaped, but are different in size from each other (size in the layer thickness direction and/or size in the wave line extending direction), and grooves are formed between adjacent projections 11A. The acellular matrix layer 1B has a plurality of projections 21A, and the projections 21A are mountain-shaped, but have different sizes from each other, and grooves are formed between adjacent projections 21A. The projections and recesses between the acellular matrix layer 1A and the acellular matrix layer 2A are fitted to each other, so that the bonding strength of both can be improved.
FIG. 3 is a schematic representation of the cross-sectional structure of a composite decellularized matrix material of an embodiment of the invention. Unlike fig. 1, the fitting structure shown in fig. 3 is serrated. Specifically, the acellular matrix layer 1B has a plurality of projections 11B, and the projections 11B have a triangular shape with a sharp tip. The acellular matrix layer 2B has a plurality of grooves 21B, and the grooves 21B have a triangular shape with a sharp tip. The projections 11B and the grooves 21B are fitted to each other, whereby the bonding strength between the acellular matrix layer 1B and the acellular matrix layer 2B can be improved.
FIG. 4A is a schematic diagram of an exploded structure of a composite decellularized matrix material of an embodiment of the invention; fig. 4B is a plan view of the lower acellular matrix layer of fig. 4A.
Unlike fig. 1, the projections in fig. 4A, 4B are cylindrical. Specifically, the acellular matrix layer 1C has a plurality of columnar grooves 11C, the acellular matrix layer 2C has a plurality of columnar projections 21C, and the projections 21C and the grooves 11C are fitted to each other, whereby the bonding strength between the acellular matrix layer 1C and the acellular matrix layer 2C can be improved.
As shown in fig. 4B, the plurality of projections 21C are arranged in a lattice on the surface of the acellular matrix layer 2C, and accordingly, it is understood that the plurality of grooves 11C are also arranged in a lattice.
FIG. 5A is a schematic diagram of an exploded structure of a composite decellularized matrix material of an embodiment of the invention; FIG. 5B is a schematic illustration of the cross-sectional structure of the composite decellularized matrix material of FIG. 5A.
Unlike fig. 4, the heights of the cylindrical protrusions shown in fig. 5A and 5B are different. Specifically, the acellular matrix layer 1D has a plurality of grooves 11D and a plurality of grooves 12D, the grooves 11D and the grooves 12D being staggered, and the depths of the two being different. In addition, the acellular matrix layer 2D has a plurality of protrusions 21D and a plurality of protrusions 22D, the protrusions 21D and the protrusions 22D are staggered, and the protrusion heights of the two are different. The projections 21D and the grooves 11D are fitted to each other, and the projections 22D and the grooves 12D are fitted to each other, whereby the bonding strength between the acellular matrix layer 1D and the acellular matrix layer 2D can be improved.
FIG. 6 is a schematic representation of the cross-sectional structure of a composite decellularized matrix material of an embodiment of the invention. Unlike fig. 4, the projections and grooves shown in fig. 6 are trapezoidal in shape, and have a shape in which the root portion is small and the tip portion is large for the projections, and a shape in which the bottom portion is large and the opening portion is small for the grooves. Specifically, the acellular matrix layer 1E has a plurality of grooves 11E, the acellular matrix layer 2E has a plurality of protrusions 21E, the protrusions 21E have a shape with a small root and a large top, the grooves 11E have a shape with a large bottom and a small opening, and the protrusions 21E and the grooves 11E are fitted to each other, so that the bonding strength between the acellular matrix layer 1E and the acellular matrix layer 2E can be improved.
FIG. 7 is a schematic representation of the cross-sectional structure of a composite decellularized matrix material of an embodiment of the invention. The difference from fig. 1 to 6 is that the composite decellularized matrix material has two decellularized matrix layers, and three decellularized matrix layers are illustrated in fig. 1 to 6. Specifically, as shown in fig. 7, the acellular matrix layers 1F, 2F, and 3F are arranged in this order, and the acellular matrix layers 1F and 2F and the acellular matrix layers 2F and 3F are all engaged with each other by the same wavy concave-convex structure as in fig. 1.
FIG. 8 is a schematic representation of the cross-sectional structure of a composite decellularized matrix material of an embodiment of the invention. Unlike fig. 7, the chimeric structure between acellular matrix layers is different. Specifically, as shown in fig. 8, the acellular matrix layer 1G and the acellular matrix layer 2G are fitted together by a zigzag uneven structure, and the acellular matrix layer 2G and the acellular matrix layer 3G are fitted together by a wavy uneven structure.
FIG. 9 is a schematic representation of the cross-sectional structure of a composite decellularized matrix material of an embodiment of the invention. As shown in fig. 9, acellular matrix layers 1H and 2H are fitted together by a zigzag uneven structure, and acellular matrix layers 2H and 3H are fitted together by a trapezoid uneven structure.
It will be appreciated that the composite acellular matrix material may also have more layers.
Example 1:
this example examines the effect of different thickening modes on decellularized matrix mechanics.
The preparation method of the composite acellular matrix material comprises the following steps:
preparing tilapia skin acellular matrix. Collecting 2 pieces of tilapia skin, removing impurities such as fish scales, peeling off the harvested dermis, washing the acellular matrix (sometimes also simply referred to as matrix) with potassium hydroxide and phosphate buffer, and cutting into square shapes. Repeatedly freezing and thawing the dermis matrix for 5 times, then treating with sodium dodecyl sulfate solution, and cleaning with purified water to obtain the fishskin acellular matrix. A small amount of citric acid is sucked by a dropper and is dripped on the surface of the acellular matrix, and the acellular matrix is uniformly smeared and then stands for reaction for a period of time. After the reaction, the substrate is washed by purified water, citric acid is continuously dripped into the other surface of one substrate for surface treatment, and the substrate is washed again after the reaction is completed. The surface treated substrate surface is a rough surface, at this time, both surfaces of one substrate are rough surfaces, and the other substrate has only one rough surface. The rough surfaces of two acellular matrixes are respectively stamped by using a wavy mould, then the two matrixes are attached, so that concave-convex parts are mutually embedded without gaps, and the schematic diagram is shown in figure 1. Two substrates were fixed with a splint and placed in a mixture of glutaraldehyde and ethanol for one day. The matrix is then expanded in water. Freeze-drying, packaging and irradiation sterilizing to obtain the final product.
Comparative example 1.1: in this comparative example, the concave-convex surface of the acellular matrix was not produced by using a mold, and only two substrates were simply bonded during the test, and the other preparation methods were the same as in example 1.
Comparative example 1.2: the method does not adopt a thickening process, and only a single piece of acellular matrix is measured during the test. The preparation of the monolithic acellular matrix was identical to that of example 1.
The acellular matrix of example 1 and comparative example 1.1 and comparative example 1.2 was cut into 5X 1cm strips, one end of which was clamped by a mechanical laboratory machine to a width of about 0.8cm, and the other end was passed through a width of 5mm from the edge with a suture, and the moving speed of the upper clamping jig was set to 60mm/min until the acellular matrix was broken. The suture stretch record is shown in detail in fig. 10. The result shows that the stress of the acellular matrix is single-piece stress, and the acellular matrix is broken one by one when broken, so that the two matrixes do not have a mechanical superposition effect; the composite acellular matrix material of the embodiment 1 has the advantages that the two substrates are tightly embedded, the suture strength of the composite acellular matrix material is 2 times that of a single substrate, and the mechanical superposition effect is achieved.
Example 2:
this example examines the effect of surface treatment and embossing to increase contact surface area on the stability of the layered decellularized matrix in solution.
The preparation method of the composite acellular matrix material comprises the following steps:
preparing salmon skin acellular matrix. Collecting 2 pieces of salmon skin, removing impurities such as fish scales, peeling off the harvested dermis, cleaning the substrate with a mixed solution of sodium hydroxide, sodium chloride and hydrogen peroxide, and cutting the substrate into square shapes. Repeatedly freezing and thawing the dermis matrix for 5 times, then treating with trypsin solution, and cleaning with purified water to obtain the fishskin acellular matrix. A small amount of acetic acid is sucked by a dropper and is dripped on the surface of the acellular matrix, and the acellular matrix is uniformly smeared and then stands for reaction for a period of time. After the reaction, the substrate is washed by purified water, citric acid is continuously dripped into the other surface of one substrate for surface treatment, and the substrate is washed again after the reaction is completed. The surface treated substrate surface is a rough surface, at this time, both surfaces of one substrate are rough surfaces, and the other substrate has only one rough surface. The rough surfaces of two acellular matrixes are respectively stamped by using a wavy mould, then the two matrixes are attached, so that concave-convex parts are mutually embedded without gaps, and the schematic diagram is shown in figure 2. Two pieces of matrix were fixed with a splint and placed in a mixture of carbodiimide and acetone for one day. The matrix is then expanded by placing it in physiological saline. Freeze-drying, packaging and irradiation sterilizing to obtain the final product.
Comparative example 2.1: the rough surface of the substrate was produced without surface treatment, and the other methods were the same as in example 2.
Comparative example 2.2: instead of using embossing to increase the contact surface area of the two substrates, only two decellularized substrates were simply attached and the rest of the process was identical to example 2.
Comparative example 2.3: the surface area increasing process by surface treatment and embossing was not used, and only two pieces of decellularized matrix were simply attached after decellularization, and the rest of the process was identical to example 2.
The acellular matrix of example 2, comparative example 2.1, comparative example 2.2 and comparative example 2.3 was immersed in water for 1 to 168 hours, and the layered acellular matrix was stirred to observe whether or not the sheet separation occurred by the observation time point. The test results are shown in Table 1. The results show that the lamination acellular matrix with the contact surface area increased by surface treatment and embossing is quickly fragmented after soaking for 1 h; the surface treatment can effectively prolong the slicing time, but the lifting time is limited; the impact of the increased contact area of stamping on the lifting and slicing time is larger, and the slicing time can be prolonged to 48 hours; the laminated acellular matrix subjected to the surface treatment and embossing surface area increasing process has the highest stability in water, does not generate a slicing phenomenon within 168 hours, and can provide mechanical support for a long time in practical application.
TABLE 1 layered decellularized matrix in water fragmentation
Note that: -indicating that the layered acellular matrix is not segmented, + indicating that the layered acellular matrix is segmented
Example 3:
this example examined the increase in thickness of the composite acellular matrix material compared to a single piece of acellular matrix.
The preparation method of the composite acellular matrix material comprises the following steps:
preparing the pigskin acellular matrix. Collecting pigskin, dehairing, peeling off the harvested dermis, cleaning the substrate with isopropanol, and cutting into square shapes. The dermal matrix is treated with sodium hydroxide and sodium dodecyl sulfate solution, and purified water is used for washing to obtain the pigskin acellular matrix. A small amount of citric acid is sucked by a dropper and is dripped on the surface of the acellular matrix, and the acellular matrix is uniformly smeared and then stands for reaction for a period of time. After the reaction, the substrate is washed by purified water, hydrochloric acid is continuously dripped on the other surface of one substrate for surface treatment, and the substrate is washed again after the reaction is completed. The surface of the substrate after surface treatment is a rough surface, and both surfaces of the substrate in the middle are rough surfaces during lamination. And respectively stamping rough surfaces of the acellular matrixes by using a wavy mould, and then attaching a plurality of matrixes to ensure that concave-convex parts are mutually embedded without gaps to prepare laminated acellular matrixes of 1 layer, 2 layers, 3 layers and 4 layers. The laminated substrate was held with a splint and fixed in ethanol and formaldehyde for one day. The matrix is then expanded in water. Freeze-drying, packaging and irradiation sterilizing to obtain the final product.
The thickness of the substrate was measured by a thickness measuring instrument at any of 10 different points on the substrate, and the average value was taken as the final thickness of the substrate. The thickness of the different stacks is detailed in table 2. As can be seen from the table, the thickness of the decellularized matrix increased with increasing number of laminated sheets. The results show that the composite acellular matrix material prepared by the invention can increase the thickness of the final acellular matrix through different lamination numbers.
TABLE 2 thickness of substrate after lamination
| Lamination number | 1 | 2 | 3 | 4 |
| Thickness (mm) | 1.07 | 1.73 | 2.48 | 3.11 |
Example 4:
this example examined the effect of surface treatment on cell adhesion.
The dermal matrix after decellularization of example 3 was selected as a material, and surface treatment was performed as in example 3. The samples after surface treatment were the samples, and the controls without surface treatment. Cutting two groups of acellular matrixes into the same size, putting the same size into a 12-hole plate, adding a complete culture medium, and culturing in an incubator for 1h until the acellular matrixes are full of the culture medium. Mouse fibroblast L929 in logarithmic growth is diluted into cell suspension according to the inoculation amount of 1×106, the cell suspension is dripped on a decellularized matrix drop by drop, static inoculation is carried out for 1h under the condition of 37 ℃ and 5% CO2, and then the cell suspension is transferred to a constant temperature orbit oscillator for dynamic rotary sowing, and the cell suspension is cultured in an incubator for 1, 3, 5 and 7 days. After each time point, the culture medium was aspirated from each set of wells, washed 3 times with PBS, and incubated in an incubator for 1-2h in the absence of light, with the addition of 2mL of a CCK-8 medium mixture. 100. Mu.L of the medium mixture was pipetted into a 96-well plate for cell proliferation assay at 450nm wavelength on a microplate reader.
The test results are shown in FIG. 11. The results show that the OD values of both groups of substrates increased with prolonged incubation time, but the OD values of the surface-treated sample groups were higher, i.e. the cell adhesion and proliferation promoting effects were stronger, than those of the control group.
The term "comprising" as used throughout this application should not be construed as limited to what is listed thereafter; it does not exclude other elements or steps. Thus, it should be construed as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more other features, integers, steps or components, or groups thereof.
It will be appreciated that those skilled in the art may implement the application in any suitable manner combining features of one or more embodiments mentioned throughout the application with features of other embodiments.
Note that the above is only the preferred embodiments of the present application and the technical principles applied. Those skilled in the art will appreciate that the present application is not limited to the particular embodiments described herein, but is capable of numerous obvious changes, rearrangements and substitutions as will now become apparent to those skilled in the art without departing from the scope of the present application. Therefore, although the present application has been described in more detail through the above embodiments, the present application is not limited to the above embodiments, and may include many other equivalent embodiments without departing from the technical concept of the present application, which falls within the protection scope of the present application.
Claims (24)
1. The preparation method of the composite acellular matrix material is characterized by comprising the following steps of:
a surface treatment step of surface roughening the surface of the first acellular matrix and surface roughening the surface of the second acellular matrix;
a fitting portion forming step of forming a first fitting portion on the surface of the first acellular matrix subjected to the surface roughening treatment and a second fitting portion on the surface of the second acellular matrix subjected to the surface roughening treatment by imprinting with a template after the surface treatment step, wherein one of the first fitting portion and the second fitting portion includes a protrusion and the other includes a groove;
a lamination step of laminating the first acellular matrix and the second acellular matrix in a state in which the first fitting portion and the second fitting portion are fitted to each other, thereby obtaining a laminate;
a fixing step of crosslinking the laminate in a state in which the laminate is sandwiched by sandwiching plates;
an expansion step of immersing the laminate in an expansion liquid after the fixing step,
a composite acellular matrix material is obtained from the laminate.
2. The method according to claim 1, wherein in the surface treatment step, both surfaces of the first acellular matrix are subjected to surface roughening treatment so that one surface of the composite acellular matrix material is roughened.
3. The method of claim 1, wherein the template has a lattice-like or wave-like concave-convex pattern.
4. The manufacturing method according to claim 1, wherein the first fitting portion and the second fitting portion are square, circular, triangular, or trapezoidal in cross section.
5. The method according to claim 1, wherein only one surface of the second acellular matrix is subjected to surface roughening treatment in the surface treatment step.
6. The method according to claim 1, wherein the surface roughening treatment is performed by applying a chemical agent or by physical shaping.
7. The method of claim 6, wherein the chemical agent comprises an acid or an oxidizing agent, wherein the acid comprises acetic acid, hydrochloric acid, or tartaric acid, and wherein the oxidizing agent comprises hydrogen peroxide or peracetic acid.
8. The method of claim 6, wherein the physical shaping comprises laser treatment, tool etching, or rough plate polishing.
9. The method according to any one of claims 1 to 8, wherein in the fixing step, the laminate is crosslinked after dehydration.
10. The method according to any one of claims 1 to 8, wherein in the fixing step, the laminate is crosslinked using one or more of formaldehyde, glutaraldehyde, epoxide, genipin, carbodiimide, hydroxysuccinimide, hexamethylene diisocyanate, acyl azide, diphenylphosphate, and glutamine transaminase.
11. The method of any one of claims 1-8, wherein the expansion fluid comprises purified water, phosphate buffer, borate buffer, or tris hydrochloride buffer.
12. The method according to any one of claims 1 to 8, characterized by further comprising, before the surface treatment process:
and an external matrix tissue collection step of collecting the external matrix tissue of the animal from the animal, and washing to remove impurities other than the matrix.
13. The method according to claim 12, wherein the washing in the external matrix tissue collecting step includes removing fat, pigment, inorganic salt or attached matter with a washing reagent.
14. The method of claim 12, wherein the non-substrate impurities are removed using a cleaning agent comprising one or more of an alcohol comprising ethanol or isopropanol, an alkali solution comprising sodium hydroxide or potassium hydroxide solution, an acid solution comprising acetic acid or citric acid, a salt solution comprising sodium chloride or potassium chloride, an oxidizing agent comprising hydrogen peroxide or potassium permanganate, an oxidizing agent comprising activated carbon or a macroporous resin.
15. The method according to claim 12, wherein in the outer matrix tissue collecting step, the outer matrix tissue is cut into a circular shape, a rectangular shape, a trapezoid shape or a hollowed-out circular shape.
16. The method according to any one of claims 1 to 8, characterized by further comprising, before the surface treatment process: a decellularized matrix preparation step of performing decellularized treatment on an external matrix tissue to obtain the first decellularized matrix and the second decellularized matrix,
In the process of preparing the acellular matrix, physical method or chemical reagent method is adopted to perform acellular treatment on the outer matrix tissue,
the physical method comprises ultrasonic wave, freeze thawing, mechanical friction, mechanical stirring or pressurizing,
the chemical reagent method uses reagents including salt solution, acid, alkali, oxidant, surfactant, and reagent capable of destroying cell membrane or binding DNA in enzyme.
17. The method of any one of claims 1 to 8, wherein the first decellularized matrix and/or the second decellularized matrix is derived from a human or an animal, including a terrestrial mammal or an aquatic animal,
the terrestrial mammal comprises pig, cow, sheep or horse, the aquatic animal comprises teleosts, cartilaginous fish or aquatic mollusk,
the first acellular matrix and/or the second acellular matrix comprises acellular dermal matrix, acellular organ membrane or acellular bone matrix.
18. A composite acellular matrix material according to any one of the preparation methods of claim 1-17, comprising a first acellular matrix layer and a second acellular matrix layer,
a first fitting portion is formed on a surface of the first acellular matrix layer facing the second acellular matrix layer,
A second fitting portion is formed on a surface of the second acellular matrix layer facing the first acellular matrix layer,
the first fitting portion is fitted to the second fitting portion, one of the first fitting portion and the second fitting portion includes a projection, and the other one includes a recess.
19. The composite acellular matrix material according to claim 18, wherein there are a plurality of the first and second fitting portions, the plurality of first and second fitting portions each having the same or different dimensions in the layer thickness direction and/or in a direction perpendicular to the layer thickness direction.
20. The composite decellularized matrix material of claim 18, wherein the first and second engagement portions are a plurality of, and wherein the first and second engagement portions are each the same or different in shape.
21. The composite decellularized matrix material of claim 18, wherein there are a plurality of said first and second engaging portions, each of said first and second engaging portions being arranged in a wavy, zig-zag or square configuration when viewed in cross-section.
22. The composite acellular matrix material according to any one of claims 18-21, further comprising a third acellular matrix layer, wherein the second acellular matrix layer is disposed between the third acellular matrix layer and the first acellular matrix layer,
A third fitting portion is formed on a surface of the third acellular matrix layer facing the second acellular matrix layer,
a fourth fitting portion is formed on a surface of the second acellular matrix layer facing the third acellular matrix layer,
the third fitting portion and the fourth fitting portion are fitted together, one of the third fitting portion and the fourth fitting portion includes a projection, and the other includes a recess.
23. The composite decellularized matrix material of claim 22, wherein the shape of said second chimeric portion and said fourth chimeric portion are the same or different.
24. The composite decellularized matrix material of any of claims 18-21, wherein said composite decellularized matrix material is rough on one side and smooth on the other side.
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| CN114222812A (en) * | 2019-05-14 | 2022-03-22 | 紫露草公司 | Composite biomaterial |
| CN114377206A (en) * | 2021-12-24 | 2022-04-22 | 杭州华迈医疗器械有限公司 | Preparation method of acellular matrix biological material |
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| WO2002053193A2 (en) * | 2001-01-02 | 2002-07-11 | The Charles Stark Draper Laboratory, Inc. | Tissue engineering of three-dimensional vascularized using microfabricated polymer assembly technology |
| US8298586B2 (en) * | 2009-07-22 | 2012-10-30 | Acell Inc | Variable density tissue graft composition |
| US10639398B2 (en) * | 2016-07-05 | 2020-05-05 | Lifecell Corporation | Tissue matrices incorporating multiple tissue types |
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| CN105769381A (en) * | 2015-05-26 | 2016-07-20 | 南通大学 | Biological patch for tissue damage restoration |
| CN114222812A (en) * | 2019-05-14 | 2022-03-22 | 紫露草公司 | Composite biomaterial |
| CN114377206A (en) * | 2021-12-24 | 2022-04-22 | 杭州华迈医疗器械有限公司 | Preparation method of acellular matrix biological material |
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