Self-assembly method of natural collagen decellularized matrix scaffold material

Abstract

The embodiment of the invention discloses a self-assembly method of a natural collagen decellularized matrix scaffold material. The method comprises the following steps: (1) soaking a fresh animal membrane tissue in a pretreatment solution to remove cell, fat, impure protein and DNA residues so as to obtain a first membrane material; (2) soaking the first membrane material in an ethanol solution, and drying to obtain a second membrane material; (3) soaking the second membrane material in a first soaking solution, and drying to obtain a third membrane material; (4) soaking the third membrane material in a second soaking solution, and drying to obtain a fourth membrane material; and (5) slitting, packaging and sterilizing the fourth membrane material to obtain the natural collagen decellularized matrix scaffold material. Wherein the first soaking solution / the second soaking solution contains glycerol and glycine. The self-assembly method provided by the invention has more excellent mechanical properties and enzymolysis resistance, meanwhile, the biocompatibility and implantation safety are higher, and the self-assembly method is widely applied in clinic.

Description

TECHNICAL FIELD

[0001] The embodiment of the present application relates to the technical field of functional materials, in particular to a self-assembly method of a natural collagen acellular matrix scaffold material. BACKGROUND

[0002] The extracellular matrix (ECM) is a complex network composed of macromolecular substances synthesized and secreted by various tissues and cells in the body, such as fibroblasts, mesenchymal cells and epithelial cells, which are distributed and aggregated on the cell surface and intercellular substances. ECM forms the cell skeleton of tissues and organs, supports and connects tissue structures, regulates tissue development, and forms the microenvironment required for cell growth.

[0003] Collagen is the main component of ECM, which is the most abundant protein in mammalian body, accounting for 25-30% of total protein and about 6% of body weight, and is widely distributed in connective tissue, skin, bone, visceral interstitial and muscle lumen, ligament, sclera and other parts. Type I collagen is the most common collagen, accounting for 90% of total collagen in human body and nearly 20% of total protein mass, mainly existing in skin, bone and tendon; type II collagen is the main component in cartilage and vitreous body.

[0004] ECM is used to prepare a scaffold material that maintains the natural three-dimensional structure of collagen and has an ideal pore structure. It is widely used in the field of tissue regeneration, including oral repair membrane, artificial brain membrane, degradable hernia patch, heart valve, etc. The ECM material of natural origin has low immunogenicity and similar spatial structure to human matrix after sufficient decellularization treatment. ECM is divided into two main sources according to the source: allogeneic and xenogeneic. Allogeneic ECM is taken from cadavers, which has limited sources, high price, and the risk of infectious diseases and is restricted by ethics, limiting its application. Xenogeneic ECM includes animal skin, pericardium, and small intestinal submucosa, which has a wide source and low price. After decellularization process, the antigenic components that can cause immune rejection are removed, while the three-dimensional spatial structure of ECM is completely preserved. After being implanted into the human body, it degrades within a certain period of time and plays its physical and biological functions during this period. Xenogeneic decellularized collagen matrix can be used for filling, repairing, barrier, and anti-adhesion of damaged tissues.

[0005] The general production process includes a series of processes such as mechanical treatment, solvent treatment, freeze-drying, sterilization, etc. to remove fat, remove host cells, remove host DNA residues, disinfect semi-finished products, inactivate viruses, sterilize, etc. After the above process, the product can be used for human use. The method of solvent treatment includes chemical method and enzyme method, the chemical method includes detergent, swelling agent, surfactant, degreasing agent, acid, alkali, oxidizing bleaching agent, etc. The acid method is more obvious in damaging the material, and should be used with caution; sodium dodecyl sulfate (SDS) is also easy to cause material denaturation; enzyme method is also an important method, but the process condition requires higher.

[0006] The existing ECM products on the market can meet most of the needs of clinical use in tissue isolation, tissue repair and guided tissue regeneration, but there are also the following problems:

[0007] Some products have a low thermal denaturation temperature, and in the case of fever in patients, the material is easy to denature and disintegrate, leading to treatment failure or reduced effect, and even causing infection; the difference in raw material site and process treatment affects the uniformity of material performance, including deformation, suture tearing, degradation rate, etc.; research has found that in the case of no fever in patients, the degradation time of the same material used in different individuals is quite different, and fever has a direct impact, and the disintegration time of the product is even less than a week. Especially in the application of artificial dura mater, the control of the degradation rate of the product is very important, and the release of a large amount of collagen protein in a short time will trigger an autoimmune system stress response, a large number of immune cells working, leading to the occurrence of effusion and even aseptic inflammation, resulting in high fever that does not subside, and even endangering life; in the process of hernia repair with high tension requirement, the rapid decline of the mechanical properties of the material and the rapid degradation of the material will bring great clinical risk, and even lead to surgical failure. The age of the animal source of the material also affects the in vivo degradation rate of the material. Generally speaking, the collagen matrix maturity of an immature individual is low, and the in vivo degradation rate will be faster, which is a potential risk in clinical application.

[0008] The irradiation sterilization of the product greatly affects the mechanical properties and degradation performance of the product. The moist heat denaturation temperature of the natural collagen protein decellularized matrix from livestock is usually above 44℃ before irradiation, and will decrease to 39-42℃ after irradiation, which brings great risk to clinical use. Once the patient is fever, the material may disintegrate within a few hours.

[0009] Freeze-drying is the mainstream product forming method at present. The benefits of freeze-drying are that it can preserve the natural morphology and uniform appearance to some extent; the cost is the consumption of energy and the rise of cost, which will be passed on to the consumer and social cost. In the current international environment of increasingly severe carbon emissions, reducing energy consumption through technology is a big trend, and high-energy production methods will be gradually eliminated by innovative technology. The products formed by freeze-drying have a large number of exposed amino acid terminal residues, which have a certain irritability to tissues. These reactions may not be detected in cytotoxicity and in vivo implantation tests, but may trigger autoimmune reactions, especially during the acute inflammatory period.

[0010] Xenogeneic ECM belongs to natural decellularized material. The performance of natural materials cannot fully meet the clinical needs in some applications, such as mechanical strength, thermal denaturation temperature, and degradation rate, and the material needs to be modified, including crosslinking, modification, etc. Through crosslinking, the active residues, especially the amino groups, are closed, reducing the immunogenicity of the material.

[0011] 1. Crosslinking modification research and application status

[0012] Crosslinking can be divided into chemical crosslinking and physical crosslinking. Chemical crosslinking is more commonly used in some applications, such as artificial heart valves. Chemical crosslinking usually has crosslinking agent residues or precipitation, such as glutaraldehyde residues or release, which can cause cytotoxicity problems.

[0013] Ultraviolet irradiation and gamma ray treatment are two methods of physical crosslinking, but gamma ray has the disadvantages of uneven and unstable irradiation dose, resulting in unstable and uneven crosslinking of the final material. Ultraviolet irradiation and gamma ray irradiation modification are difficult to industrialize due to poor controllability. Severe dehydration (DHT) is also a commonly used method in collagen protein physical modification. This method crosslinks collagen protein through high-temperature dehydration to improve denaturation temperature and improve the physical properties of collagen. Severe dehydration shortens the distance between active groups of collagen protein, causing crosslinking between collagen protein molecules, increasing the denaturation temperature of collagen protein, reducing the content of free amino acids, and thus improving the mechanical strength of collagen protein. Severe dehydration and thermal crosslinking modification have certain application value, but through public research data, it is found that the crosslinking degree is low, and the product performance improvement effect is small, which limits its industrialization application. And the above methods will cause a bond breakage of collagen a and partial denaturation.

[0014] 2. Improvement of physical crosslinking modification method

[0015] In the traditional DHT research process, some basic elements of materials science are ignored, such as the relationship between intermolecular forces and intermolecular distances, including the role of liquid phase systems in crosslinking.

[0016] Collagen is characterized by three polypeptides with molecular weight of more than 1000 amino acids, which are intertwined into collagen molecules (diameter of about 1.5 nm) with a triple helix structure, and multiple parallel collagen molecules are crosslinked in front, back, left and right to further aggregate into collagen structures of different levels, and finally form collagen fibers. Collagen has a quaternary structure, in which the primary structure refers to the order of amino acid connection, which is the basic structure of protein; the secondary, tertiary and quaternary structures are three-dimensional spatial structures, which are the higher structures of protein. The triple helix conformation is the basis of the physicochemical properties and biological activities of collagen, and the forces stabilizing the triple helix mainly include hydrogen bonds, van der Waals forces and covalent crosslinking bonds.

[0017] The formation of crosslinking bonds in the DHT crosslinking process depends on the complete removal of bound water in collagen molecules. The removal of bound water leads to condensation reactions of carboxyl groups and amino groups on the side chains of adjacent amino acids to form ester bonds and amide bonds, resulting in intermolecular crosslinking of collagen, which can significantly improve the mechanical properties of collagen fibers. The dehydration process of collagen fibers in a vacuum state is accompanied by simultaneous crosslinking and denaturation reactions of collagen. A proper crosslinking temperature will produce a suitable dehydration rate, making the reactions in collagen mainly crosslinking and secondarily denaturation. At a temperature of 110°C, the terminal carboxyl, amino and hydroxyl groups have increased activity due to the increase in temperature, and intermolecular copolymerization dehydration reactions of adjacent residues occur; when the collagen fibers lose intermolecular bound water under high temperature to form new chemical bonds, the collagen molecules will rearrange to different degrees. Physical crosslinking produces new chemical bonds and new charged groups, affecting the crystallization properties of collagen. The internal microfibril structure of DHT crosslinked collagen fibers is more compact, and the triple helix structure of collagen molecules in the crosslinked fibers is basically preserved.

[0018] The formation of crosslinking in the DHT treatment process depends on the degree of removal of bound water and the initial form of the material. Taking collagen protein as an example, there is a big difference in nature between the collagen protein decellularized matrix of natural structure, which will also bring different crosslinking results; even if it is a collagen protein decellularized matrix of natural structure, the final forming method will also bring differences in material properties. For example, the space structure and properties of the lyophilized product will affect the research results. If the natural collagen membrane is first dehydrated by lyophilization and then subjected to severe dehydration, the intermolecular distance will be fixed due to lyophilization, and only a small number of adjacent residues will undergo intermolecular copolymerization dehydration reaction, and the formation of hydrogen bonds and ester bonds will be less. These reactions can only occur between adjacent molecules, and the reactions between the residues in the spatial structure of collagen protein, including between collagen fibers, three-dimensional helices, primary and secondary structures, hydrogen bonds and covalent bonds, have a lower probability of occurrence.

[0019] In traditional esterification reactions, concentrated sulfuric acid protonates the carbonyl group in the carboxyl group of the carboxylic acid, forming an unstable carbocation intermediate with two hydroxyl groups. This intermediate then dehydrates to form an acyl cation. Therefore, the catalytic effect of concentrated sulfuric acid essentially utilizes its strong acidity and strong protonating ability to promote the formation of the acyl cation, lowering the activation energy and increasing the reaction rate. The hygroscopic property of concentrated sulfuric acid does not have a catalytic effect, but rather acts to alter the equilibrium, causing the esterification reaction to proceed in the forward direction and reducing the reverse reaction of ester hydrolysis.

[0020] Our research revealed that altering the distance between collagen amino acid residues (active groups) is crucial for self-assembly (self-crosslinking). This change in the distance between collagen residues cannot be achieved through simple material compression, but can be accomplished through saturated water absorption followed by drying. However, this finding currently only applies to natural decellularized collagen matrix materials; sufficient self-crosslinking has not yet been observed in macromolecular collagen materials.

[0021] Taking mainstream freeze-dried collagen membrane products as an example, the amino acid residues in the membrane product are in a spread-out dissolved state in aqueous solution. The carboxyl group also has a hydroxyl group. The oxygen on this hydroxyl group can form a hydrogen bond with a hydrogen atom in water, and the hydrogen on this hydroxyl group can form a hydrogen bond with an oxygen atom in a water molecule. Therefore, in principle, one carboxyl group can form three hydrogen bonds with a water molecule. Through rapid freezing and freeze-drying, this state is basically maintained. During the vaporization dehydration process, the positions of the amino acid residues are fixed, and most residues are separated by distances greater than intermolecular forces, so they do not interact during dehydration. After freeze-drying, the terminal carboxyl, amino, and hydroxyl groups are in a dehydrated, curled state, hence the product appears white.

[0022] By controlling the self-assembly process of collagen membranes, the intermolecular distance can be controlled. During collagen membrane self-assembly, continuous dehydration leads to a continuous reduction in the intermolecular distance, resulting in highly efficient hydrogen bonding and covalent bond reactions. In this process, amino acid residues are initially in an extended state due to the presence of water. When two adjacent molecules approach each other and are both in an ionized state, the reaction occurs very easily, and the hydrogen bond distance is typically between 0.15 and 0.35 nanometers. When the hydrogen bond distance is less than 0.15 nanometers, the hydrogen bond energy becomes very strong, leading to a compact molecular structure. When the hydrogen bond distance is greater than 0.35 nanometers, the force decreases significantly. Near the end of water evaporation, water molecules pull two adjacent residues to a very close distance or into contact, reducing the intermolecular distance to less than 0.35 nanometers. The molecules of the two residues are then connected by strong forces through hydrogen bonding or covalent bonding. The natural dehydration process, with its loss of moisture, reduces the spatial support capacity of collagen. Simultaneously, hydrogen and covalent bond reactions cause the collagen membrane to collapse and shrink, with the shrinkage rate in the vertical direction reaching 1 / 3 to 1 / 5 of the saturated water-absorbing state—that is, a change in membrane thickness. This change is irreversible. We conducted the following experiment: a 2-3% collagen solution, after drying to form a film, disintegrated after approximately 24 hours of rehydration, dispersing into a gel-like state in water. In contrast, a natural collagen membrane, after drying and rehydration for up to 30 days, showed decreased transparency, but its thickness remained essentially the same as after self-assembly, not returning to its pre-drying state. This suggests that, in addition to hydrogen bonding, esterification or amidation reactions also occurred. This reaction has been well demonstrated in previous DHT studies. Unlike high-temperature and vacuum reactions, self-assembly reactions at near-room temperature are very easy to achieve. Collagen macromolecular dried films are difficult to maintain their morphology in water, which may be due to two reasons: First, the hydrophobic forces or other forces in the natural collagen structure mediate intermolecular self-crosslinking reactions, promoting a large number of reactions; second, in the near-room temperature severe dehydration reaction of collagen peptides, hydrogen bonds are the main component, and self-crosslinking reactions occur less frequently, or a small number of local self-crosslinking reactions occur, but they cannot maintain a stable spatial structure.

[0023] This water-mediated collagen self-assembly behavior differs from the cross-linking process mediated by chemical catalysts / dehydrating agents that we usually understand; it is also different from the heavy dehydration / DHT method. Conventional heavy dehydration of natural collagen membranes mainly involves dry materials, resulting in large spatial distances between residues. The low water content and the non-ionization and ionization activation state of collagen active groups also limit their self-assembly. In the self-assembly process, water acts as both a temporary filler and a temporary activator, providing the necessary conditions for the reaction of membrane protein residues through a near-natural evaporation process. During collagen dehydration, the collagen membrane material continuously shrinks and thins due to cross-linking reactions. Simultaneously, previously distant amino acid residues move closer together, initiating new reactions. These cross-linking reactions can occur within peptide bonds, between three-dimensional helices, and between collagen fibers. These reactions further shrink the collagen matrix, making the material more compact. The force generated per unit time during the cross-linking and shrinkage process is relatively small, less than the frictional force between the material and the carrier. Therefore, the material thickness decreases significantly, but the material area does not change substantially. The surface tension of water also plays a crucial role in this process. It can be imagined that as the material approaches dryness, the water between collagen fibers evaporates first. The water at the amino acid ends is the most difficult to remove because amino acid ends are strongly hydrophilic. Adjacent residues share water at the last moment and are connected due to the surface tension of the water. As the water evaporates, two ionized residues react at extremely close range at the instant of water removal, such as esterification and amidation reactions. This differs from reactions typically mediated by various conditions; it is a chemical bond reaction close to its natural state.

[0024] The demacrolysis and decellularization process of collagen affects the exposure of amino acid residues in the material. The process uses a large amount of strong oxidants and strong bases, which helps to hydrolyze a large number of amide bonds in an alkaline manner, forming carboxylic acid and amino residues. Without affecting the structural stability of the material, this process is conducive to the occurrence of water-mediated self-assembly reaction under water-deficient conditions.

[0025] The amino acid residues of natural collagen scaffolds exhibit strong reactivity. During stepwise dehydration, numerous intermolecular interactions occur, which can be considered a broad self-assembly process (including self-crosslinking) involving hydrogen bonds, ester bonds, and amide bonds. Some weaker reactions (hydrogen bonds) gradually dissociate during rehydration, but a large portion of these reactions (ester and amide bonds) are irreversible under normal conditions, preventing the collagen membrane thickness and morphology from returning to their pre-dehydration state. We conducted long-term observations of the self-assembled product. Over a period exceeding one month, the material's thickness in phosphate buffer did not change significantly, but the material rapidly changed from translucent to white, confirming our conclusions.

[0026] By controlling the dehydration rate, the membrane self-assembly process can be controlled, thereby controlling the sufficiency of membrane material reaction. The thickness, tensile strength, elongation, and residue reaction rate of the membrane material can be controlled simultaneously.

[0027] Ethanol can denature proteins primarily through the following mechanisms: 1. Ethanol can provide hydrogen or oxygen from its hydroxyl or carbonyl groups to form hydrogen bonds, thereby disrupting the existing hydrogen bonds in the protein and causing denaturation. However, hydrogen bonds are not chemical bonds, so there is no breaking or formation of chemical bonds during the process; it is a physical change and is often reversible. In practical applications, ethanol denatures proteins mainly through the second pathway: ethanol disrupts the hydration layer on the protein surface. The hydration layer consists of polar groups on the protein surface that adsorb water molecules, forming an ordered hydration layer. This hydration layer encapsulates the protein, allowing it to dissolve in water and maintain its biological activity. If the hydration layer is disrupted, the environment upon which the protein depends for survival changes, leading to protein denaturation.

[0028] This principle can be used to pretreat products with ethanol, thereby controlling the extent of hydrogen bonding reactions in the material. By adding certain non-aqueous components to the aqueous phase, the efficiency of self-assembly can be controlled to some extent. Within a certain range, the higher the content of non-aqueous components, the less hydration layer of collagen, and the lower the self-assembly reaction rate. The sufficiency of the self-assembly reaction can be controlled in this way.

[0029] Glycine, as a whole, is a polar molecule (all amino acids are polar), but it is classified as a nonpolar amino acid. This is because the polarity of an amino acid is determined by the nature of its R group, not the entire molecule. The glycine chain has a hydrogen atom in each branch, classifying it as a hydrocarbon chain and thus nonpolar. Similarly, although it is readily soluble in water, it is a hydrophobic amino acid. However, in biological systems, it is generally classified as nonpolar.

[0030] The glycine blocking principle is a commonly used chemical synthesis method for synthesizing biomolecules such as peptides and proteins. Glycine is an amino acid with both hydrophobic and hydrophilic properties, which allows it to act as a barrier in chemical reactions. In the synthesis of peptides or proteins, amino acid molecules need to be linked together one by one to form a peptide chain. However, because the carboxyl and amino groups in amino acid molecules readily react, this can lead to incomplete or broken peptide chains. The glycine blocking principle can be used to address this problem. Both the carboxyl and amino groups in glycine molecules can react with other amino acid molecules.

[0031] Similar to the principle of dehydration-induced self-assembly, adding a certain concentration of glycine to a collagen membrane soaking solution during gradual dehydration causes two free residues in glycine to undergo hydrogen / covalent bond reactions similar to collagen membrane self-assembly. This process is not typical self-assembly, but glycine is the most abundant amino acid in collagen, and the results show that it does not affect the basic structure and properties of collagen. However, it is foreseeable that different glycine contents and different reaction conditions, including ionic strength, temperature, and reaction time, will affect the degree of self-assembly. By externally adding glycine, the self-assembly behavior of materials can be controlled to some extent. Based on the above analysis, we designed an experiment: two decellularized collagen membrane materials of equal weight were each added with 0.5% glycine self-assembly buffer until they were nearly saturated. One sample (Sample 1) was stored in a sealed bag at 0-8℃, while the other (Sample 2) was assembled using the self-assembly method (35℃, 2 hours). After immersion in purified water for 12 hours under the same conditions, the glycine content in the extract of Sample 1 was measured to be 0.12%, and the glycine content in the extract of Sample 2 was measured to be 0.02%. This experiment shows that the free glycine content in the material decreased significantly after self-assembly, suggesting that the added glycine also underwent a self-assembly reaction. Glycine blocking has positive effects on the modification and immunogenicity control of natural collagen and is a good supplement to dehydration self-assembly; however, the specific extent of its impact requires further research. Summary of the Invention

[0032] Therefore, embodiments of the present invention provide a method for self-assembly of a natural collagen decellularized matrix scaffold material.

[0033] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0034] According to a first aspect of the present invention, the present invention provides a method for self-assembly of a decellularized natural collagen matrix scaffold material, the method comprising the following steps:

[0035] (1) Fresh animal membrane tissue was immersed in a pretreatment solution to remove cells, fat, impurities, proteins, and DNA residues, and the first membrane material was obtained.

[0036] (2) The first membrane material is immersed in an ethanol solution and dried to obtain the second membrane material;

[0037] (3) Immerse the second membrane material in the first immersion solution and dry it to obtain the third membrane material;

[0038] (4) Immerse the third membrane material in the second soaking solution, dry it, and then prepare the fourth membrane material;

[0039] (5) The fourth membrane material is cut, packaged, and sterilized to obtain the natural collagen decellularized matrix scaffold material;

[0040] The first soaking solution / second soaking solution contains glycerol and glycine.

[0041] Further, the first soaking solution / second soaking solution comprises, by volume concentration: 2-20 g of glycerol / 100 ml, 0.1-0.5 g of glycine / 100 ml, and the balance being a buffer solution with pH = 5-8.

[0042] Further, in step (1), the pretreatment solution comprises, by weight percentage: 0.5-10% alkali, 1-10% oxide, 0.5-5% degreasing agent, and the balance being water; wherein the alkali is sodium hydroxide, potassium hydroxide, sodium carbonate, trisodium phosphate, sodium tripolyphosphate, or sodium pyrophosphate, the oxide is hydrogen peroxide, and the degreasing agent is Tween 80, Triton X-100, or fatty alcohol polyoxyethylene ether AEO-9; the soaking temperature is 0-25℃, and the soaking time is 20-60 hours. To improve the pretreatment effect and shorten the soaking time, the pretreatment solution can be replaced during the soaking process.

[0043] Further, in step (2), the concentration of the ethanol solution is 50-90%; the soaking temperature is 0-10℃ and the time is 1-5h; the drying temperature is 30-40℃ and the time is 0.5-5h.

[0044] Further, in step (3), the soaking temperature is 0-10℃ and the time is 0.5-5h; the drying temperature is 30-40℃ and the time is 0.5-5h.

[0045] Further, in step (4), the soaking temperature is 10-25℃ and the time is 0.5-5h; the drying temperature is 30-55℃ and the time is 0.5-5h.

[0046] Further, the concentration of the buffer solution is 0.05-0.5 mol / L, and the buffer solution is a phosphate buffer and / or a citrate buffer; the phosphate includes phosphoric acid, sodium phosphate, and potassium phosphate; the citrate includes citric acid, sodium citrate, and potassium citrate.

[0047] According to a second aspect of the present invention, the present invention provides a natural collagen decellularized matrix scaffold material, which is made by the method described in any of the preceding claims.

[0048] The embodiments of the present invention have the following advantages:

[0049] 1. The support material of this invention is formed and processed by a multi-step self-assembly method, which can solve the problem of high energy consumption caused by freeze drying, significantly reduce production costs, and is more socially and environmentally friendly.

[0050] 2. The scaffold material of the present invention is formed and processed by a multi-step self-assembly method, which can appropriately seal the amino acid terminal residues of the product, thereby improving biocompatibility and implantation safety.

[0051] 3. The stent material of the present invention is formed by a multi-step self-assembly method, and the material forming thickness can be controlled within a certain range, which can better adapt to different clinical needs.

[0052] 4. The stent material of this invention is formed by a multi-step self-assembly method, which greatly improves the mechanical properties of the material and can meet the repair needs of high-requirement sites in clinical practice.

[0053] 5. The scaffold material of this invention is formed and processed by a multi-step self-assembly method, which significantly improves the product's resistance to enzymatic hydrolysis and can better guarantee the degradation time and degradation rate.

[0054] 6. The stent material of this invention is formed by a multi-step self-assembly method, and the elongation at break and deformation of some special materials are significantly reduced, making it more ideal for clinical application.

[0055] 7. The glycine-blocked terminal residues in the scaffold material of this invention reduce the immunogenicity of the material and can control the self-assembly process of the material to a certain extent.

[0056] 8. The scaffold material of the present invention is formed by self-assembly, which can achieve product performance close to that of chemical cross-linking, without introducing cross-linking agent residue, while retaining more amino acid residues, and has better activity in guiding tissue regeneration. Attached Figure Description

[0057] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0058] Figure 1 Rehydration diagrams of freeze-dried bovine pericardium sample (left) and self-assembled sample (right, processed by self-assembly process in Example 1);

[0059] Figure 2 Thermal shrinkage images of freeze-dried bovine pericardium sample (left) and self-assembled sample (right, processed by self-assembly process in Example 1) (same raw material, 61°C). Detailed Implementation

[0060] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0061] Example 1

[0062] Prepare the solution:

[0063] Pretreatment solution: Mix 5g sodium hydroxide, 20g hydrogen peroxide, 95g AOE-95g and 970g water until homogeneous.

[0064] Soaking solution: Add 40g of glycerol and 1g of glycine to 200ml of 0.5mol / L sodium phosphate / sodium dihydrogen phosphate buffer solution and stir until well mixed.

[0065] This embodiment provides a self-assembly method for a decellularized matrix scaffold material made of natural collagen:

[0066] (1) Take fresh beef heart, manually remove the fascia components, and soak it in a pretreatment solution at 20-25℃ for 20 hours to obtain decellularized collagen membrane.

[0067] (2) Place the membrane material obtained in step (1) in a 50% ethanol solution, soak it at 6°C for 2 hours, take it out, and dry it at 30°C for 1 hour.

[0068] (3) Place the membrane material obtained in step (2) in the soaking solution, soak it at 8°C for 1 hour, take it out, and dry it at 30°C for 2 hours to obtain a relatively soft collagen membrane. Most areas are mixed with some air bubbles, and some areas are white. The material has poor appearance uniformity.

[0069] (4) Place the membrane material obtained in step (3) in the soaking solution, soak it at 20°C for 1 hour, take it out, and dry it at 30°C for 2 hours to obtain a semi-transparent soft collagen membrane. The membrane material has a smooth and firm appearance.

[0070] (5) The product is cut, double-layered, and sterilized with ethylene oxide.

[0071] The appearance, rehydration performance and mechanical properties (tensile strength) of the membrane materials obtained in steps (1)-(4) were tested, and the results are shown in Table 1.

[0072] The tensile strength test method involves randomly selecting samples from each stage, rehydrating them, cutting them into 10mm × 4mm rectangles, and measuring their average thickness with calipers. The samples are then tested using a tensile testing machine, and the maximum tensile force at fracture is recorded.

[0073] Test method for rehydration performance: Place the material in pure water and record the time it takes for the material to fully soften.

[0074] Table 1

[0075]

[0076] Example 2

[0077] Prepare the solution:

[0078] Pretreatment solution: Mix 10g sodium carbonate, 50g hydrogen peroxide, 8g Tween 80 and 932g water until homogeneous.

[0079] Soaking solution: Add 40g of glycerol and 0.5g of glycine to 200ml of 0.5mol / L sodium phosphate / sodium dihydrogen phosphate buffer solution and stir until well mixed.

[0080] This embodiment provides a self-assembly method for a decellularized matrix scaffold material made of natural collagen:

[0081] (1) Take fresh cowhide, remove hair and subcutaneous fat fascia, and soak it in a pretreatment solution at 15-20℃ for 40 hours, changing the solution once in between, to obtain decellularized collagen membrane.

[0082] (2) Place the membrane material obtained in step (1) in a 70% ethanol solution, soak it at 4°C for 5 hours, take it out, and dry it at 30°C for 1 hour.

[0083] (3) Place the membrane material obtained in step (2) in the soaking solution and soak it at 5°C for 1.5 hours. Take it out and dry it at 35°C for 2 hours to obtain a semi-transparent soft collagen membrane. The thickness of the membrane material decreased significantly, with some air bubbles mixed in and local whitening.

[0084] (4) Place the membrane material obtained in step (3) in the soaking solution and soak it at 20°C for 1 hour. Take it out and dry it at 40°C for 3 hours to obtain a semi-transparent collagen membrane. The membrane material has a certain degree of flexibility, and its appearance is flat, smooth and tight. The thickness is slightly lower than that after the first drying. The membrane material has a flat, smooth and tight appearance.

[0085] (5) The product is cut, double-layered, and sterilized with ethylene oxide.

[0086] The appearance, rehydration performance and mechanical properties of the membrane materials obtained in steps (1)-(4) were tested (the test methods are the same as in Example 1), and the results are shown in Table 2.

[0087] Table 2

[0088]

[0089] Example 3

[0090] Prepare the solution:

[0091] Pretreatment solution: Mix 20g of trisodium phosphate, 60g of hydrogen peroxide, 5g of Triton X-100 and 915g of water until homogeneous.

[0092] First soaking solution: Add 100g of glycerol and 2g of glycine to 500ml of 0.2mol / L sodium phosphate / sodium dihydrogen phosphate buffer solution and stir until well mixed.

[0093] Second soaking solution: Add 25g of glycerol and 1g of glycine to 500ml of 0.2mol / L sodium phosphate / sodium dihydrogen phosphate buffer solution and stir well.

[0094] This embodiment provides a self-assembly method for a decellularized matrix scaffold material made of natural collagen:

[0095] (1) Take fresh pig small intestine, remove the fat, and soak it in a pretreatment solution at 20-25℃ for 56 hours, changing the solution once in between, to obtain a decellularized collagen membrane.

[0096] (2) Place the membrane material obtained in step (1) in a 70% ethanol solution, soak it at 5°C for 2 hours, take it out, and dry it at 40°C for 1 hour.

[0097] (3) The membrane material obtained in step (2) is placed in the first soaking solution and soaked at 5°C for 2 hours. It is then taken out and dried at 30°C for 5 hours to obtain a relatively soft collagen membrane. Most areas are mixed with some air bubbles, and some areas are white. The material has poor appearance uniformity.

[0098] (4) Place the membrane material obtained in step (3) in the second soaking solution, soak it at 25°C for 1 hour, take it out, and dry it at 30°C for 5 hours to obtain a semi-transparent soft collagen membrane. The membrane material has a smooth and firm appearance.

[0099] (5) The product is cut, double-layered, and sterilized with ethylene oxide.

[0100] The appearance, rehydration performance and mechanical properties of the membrane materials obtained in steps (1)-(4) were tested, and the results are shown in Table 3.

[0101] Table 3

[0102]

[0103] Example 4

[0104] Prepare the solution:

[0105] Pretreatment solution: Mix 10g sodium carbonate, 80g hydrogen peroxide, 8g Tween 80 and 902g water until homogeneous.

[0106] First soaking solution: Add 100g of glycerol and 4g of glycine to 1000ml of 0.4mol / L disodium hydrogen phosphate / sodium dihydrogen phosphate buffer solution and stir until well mixed.

[0107] Second soaking solution: Add 60g of glycerol and 5g of glycine to 1000ml of 0.3mol / L disodium hydrogen phosphate / sodium dihydrogen phosphate buffer solution and stir until well mixed.

[0108] This embodiment provides a self-assembly method for a decellularized matrix scaffold material made of natural collagen:

[0109] (1) Take fresh pig skin, remove the grease with a degreasing machine, remove the hair with alkali solution, and trim the thickness of the skin base; at 20-25℃, soak it in the pretreatment solution for 60 hours, changing the solution 3 times in between, to obtain a decellularized collagen membrane.

[0110] (2) Place the membrane material obtained in step (1) in a 90% ethanol solution, soak it at 0°C for 5 hours, take it out, and dry it at 40°C for 5 hours.

[0111] (3) The membrane material from step (2) is placed in the first soaking solution and soaked at 5°C for 2 hours. It is then removed and dried at 40°C for 5 hours to obtain a relatively soft collagen membrane. Most areas are mixed with some air bubbles, and some areas are white. The material has poor appearance uniformity.

[0112] (4) Place the membrane material obtained in step (3) in the second soaking solution, soak it at 25°C for 1 hour, take it out, and dry it at 30°C for 3 hours to obtain a semi-transparent soft collagen membrane. The membrane material has a smooth and firm appearance.

[0113] (5) The product is cut, double-layered, and sterilized with ethylene oxide.

[0114] The appearance, rehydration performance and mechanical properties of the membrane materials obtained in steps (1)-(4) were tested, and the results are shown in Table 4.

[0115] Table 4

[0116]

[0117] Example 5

[0118] Prepare the solution:

[0119] Pretreatment solution: Mix 5g sodium tripolyphosphate, 100g hydrogen peroxide, 10g AOE-9 and 885g water until homogeneous.

[0120] First soaking solution: Add 50g of glycerol and 3g of glycine to 1000ml of 0.2mol / L sodium phosphate / sodium dihydrogen phosphate buffer solution and stir until well mixed.

[0121] Second soaking solution: Add 150g of glycerol and 4g of glycine to 2000ml of 0.3mol / L sodium phosphate / sodium dihydrogen phosphate buffer solution and stir well.

[0122] This embodiment provides a self-assembly method for a decellularized matrix scaffold material made of natural collagen:

[0123] (1) Take fresh pig pericardium, remove hair with alkaline solution, and soak it in pretreatment solution at 10-25℃ for 30 hours, changing the solution once in between, to obtain decellularized collagen membrane.

[0124] (2) Place the membrane material obtained in step (1) in a 50% ethanol solution, soak it at 5°C for 4 hours, take it out, and dry it at 35°C for 2 hours.

[0125] (3) The membrane material obtained in step (2) is placed in the first soaking solution and soaked at 8°C for 2 hours. It is then taken out and dried at 35°C for 3 hours to obtain a relatively soft collagen membrane. Most areas are mixed with some air bubbles, and some areas are white. The material has poor appearance uniformity.

[0126] (4) Place the membrane material obtained in step (3) in the second soaking solution, soak it at 20°C for 1 hour, take it out, and dry it at 40°C for 4 hours to obtain a semi-transparent soft collagen membrane. The membrane material has a smooth and firm appearance.

[0127] The appearance, rehydration performance and mechanical properties of the membrane materials obtained in steps (1)-(4) were tested, and the results are shown in Table 5.

[0128] Table 5

[0129]

[0130] ECM tissue-engineered products used clinically include heart valves, artificial dura mater, hernia patches, and implant membranes. Heart valves, artificial dura mater, and hernia patches all have chemically cross-linked products, but chemical cross-linking methods will not be discussed here. For non-chemically cross-linked ADM products already on the market, the dry products are primarily lyophilized, with most being sterilized by irradiation.

[0131] Among the known indications, ECM (extracorporeal membrane membrane) is used to repair soft and hard tissues caused by various reasons, serving as a guide, isolate, repair, fix, reduce tension, and replace membrane material with collagen as its main component. The basic requirements for ECM include mechanical strength, degradation performance, biocompatibility, and the ability to guide regeneration. Biocompatibility is the most fundamental requirement for ADM (anti-inflammatory membrane). After self-assembly, the terminal residues of ECM undergo self-crosslinking condensation, significantly reducing the material's irritation to tissues and resulting in lower immune rejection compared to lyophilized materials. Furthermore, through shrinkage, the thickness of the material is reduced to one-third or even less of that of lyophilized materials, resulting in a smaller footprint at the wound site, which also greatly reduces the material's irritation to the body.

[0132] When ECM materials are used to guide regeneration, physical properties are a basic requirement, serving as the foundation and guarantee for tissue regeneration. Taking implant membranes and meninges as examples, effective barrier isolation is the most important performance requirement. Usually, the materials are degraded and absorbed after implantation, so it is not required that autologous cells grow and multiply in large quantities within the implanted material. Guided soft tissue regeneration refers to the effect of guiding the growth of tissues (autologous tissues) on both sides of the material, and the generated soft tissue should be flat and smooth. In bone repair, bone tissue regeneration is guided by the stable formation of osteogenic spaces.

[0133] In vivo degradation is one of the most important indicators of ECM materials. The in vivo degradation of materials depends on their physical and chemical properties. Mechanical properties are an important characterization of degradation, and furthermore, intermolecular forces and chemical bonds affect the in vivo degradation performance and cycle. Studies have shown that self-assembled collagen raw materials exhibit significantly improved physical properties and a significantly longer in vivo degradation time compared to freeze-dried materials. We designed an accelerated enzymatic hydrolysis experiment to verify the effect of material self-assembly. Accelerated enzymatic hydrolysis experiment: Ten freeze-dried bovine pericardium samples and ten self-assembled bovine pericardium samples (freeze-drying was performed using a conventional low-temperature freeze-vacuum drying process; self-assembly was performed using the process in Example 1) were randomly selected, each measuring 2cm*2cm. Five samples from each sample were sterilized with Cobalt-60 at 25KGY. The weight (Wo) was measured, and the samples were immersed in 1mg / mL collagenase at 37℃. The materials were then dried at 40℃ every 2 hours, and their mass (Wt) was measured. Wt / Wo represents the enzymatic hydrolysis residue rate. The results are shown in Table 6.

[0134] Table 6

[0135]

[0136] The results showed that the self-assembled samples provided by this invention exhibited significantly improved in vitro enzymatic resistance compared to lyophilized samples. Cobalt-60 irradiation had a significant impact on the in vitro enzymatic resistance of the materials; after 25 kGy irradiation sterilization, the enzymatic resistance of the lyophilized samples decreased to almost 50% of the original sample's resistance. However, after the self-assembly process of this invention, the impact of irradiation on the samples' enzymatic resistance was relatively small. It can be inferred that if ECM materials are processed using a self-assembly combined with ethylene oxide sterilization, the material's resistance to degradation will be significantly improved.

[0137] ECM materials prepared through self-assembly exhibit higher mechanical strength and less uniform tensile deformation in all directions, further improving surgical success rates and accuracy, and better ensuring surgical outcomes. The increased thermal denaturation temperature of self-assembled ECM materials reduces the risk of short-term material disintegration and failure, and ensures more reliable in vivo degradation time, guaranteeing both short- and long-term surgical results. In clinical use, self-assembled ECM materials possess a translucent appearance and better rigidity than lyophilized products, facilitating observation and manipulation, thus bringing greater value to clinical applications. A parallel comparative study of lyophilized and air-dried products systematically investigated the transparency, maximum tensile strength, density, porosity, water saturation, in vitro degradation rate, cytotoxicity, tissue compatibility, in vivo degradation rate, and collagen arrangement of the two materials. Except for lower porosity, the air-dried product outperformed the lyophilized product in most performance indicators.

[0138] Product performance is improved through self-assembly, opening up more possibilities for the processing and application of certain clinical products. Artificial heart valves have high requirements for thickness and mechanical strength, with thickness directly affecting delivery size. If the biomembrane is too thick, its overall flexibility is poor, increasing compression difficulty and resulting in a larger overall size, making implantation at the target site difficult and potentially causing complications during delivery. If the biomembrane is too thin, its tensile strength is poor, making it prone to premature tearing. Through room-temperature self-assembly pretreatment, biomembrane materials can be pre-shaped, thickness controlled, and further chemically cross-linked, allowing for the design and control of material flexibility / rigidity. In some high-risk applications of hernia patches, chemically cross-linked materials exhibit better stability. Self-cross-linked materials possess physical properties and degradation characteristics similar to chemically cross-linked materials, providing more possibilities for clinical material selection in this field. Taking hernia patches as an example, the materials require high tensile strength and suture tear resistance, must withstand the mechanical performance requirements after implantation, and have higher degradation requirements. Currently, some non-crosslinked products are used clinically, but compared with chemically crosslinked materials, they pose higher risks during use. Chemically crosslinked products have more stable material properties, but some studies have indicated that their effect on promoting autologous tissue repair and regeneration is weaker than that of non-crosslinked products. Self-assembled (crosslinked) materials have relatively weaker amino acid residue sealing compared to chemically crosslinked materials, but they have significantly improved material strength and degradation resistance compared to non-crosslinked products, combining the advantages of both non-crosslinked and chemically crosslinked products. This innovative technology can solve the limitations of material performance in clinical applications and has significant clinical implications. Taking the submucosa of the small intestine as an example, single-layer SIS has the highest elongation, reaching approximately 130%, while multilayer SIS has an elongation of 30%–40%. Furthermore, single-layer SIS is anisotropic, which limits the application of its single-layer products. The mechanical strength varies greatly in different parts of decellularized porcine skin, and the abdominal matrix is ​​very loose, with a fracture elongation exceeding 100%, causing great inconvenience in application. Self-assembly can greatly improve these characteristics. We conducted the following experiments using pig skin abdominal samples: cells were removed using a decellularization process, and the samples were treated with two methods: freeze-drying and self-assembly (freeze-drying was performed using a conventional low-temperature freeze-vacuum drying process; self-assembly was performed using Example 4). The samples were then sterilized by 25 kgy irradiation. Three samples of each method were taken and stretched using a thin film tensile testing machine at a test speed of 1 mm / min until the deformation reached 0.125 mm, and then stretched at 100 mm / min until the film broke. The results are shown in Table 7.

[0139] Table 7

[0140] Sample / Stretch Ratio 1 2 3 Average Lyophilized 25KGY 105％ 92％ 95％ 97.3％ Self-assembly 25KGY 55％ 63％ 58％ 58.7％

[0141] The results above show that the deformation of samples obtained by the self-assembly process is greatly reduced, providing a more ideal solution for the processing of some special raw materials.

[0142] To achieve better self-assembly results, a multi-step self-assembly method was designed, and glycerol was introduced into the self-assembly solution. Glycerol, as a solvent, is non-volatile below 130℃ and has a boiling point of 290℃. During the self-assembly process, non-ionizing barriers are formed between some neighboring molecules, controlling the occurrence of the self-assembly reaction and thus controlling the progress / degree of the self-assembly reaction. Introducing glycine to block the terminal residues of amino acids can further enhance / complement the self-assembly process.

[0143] Take decellularized cowhide as an example:

[0144] The membrane material was treated with a strong alkali and enhanced oxidant process. It was degreased, deproteinized, and had its DNA residue removed. It was then freeze-dried / dehydrated and self-assembled. The resulting material was observed and measured. The results are shown in Table 8.

[0145] Table 8

[0146]

[0147] Taking decellularized pigskin as an example:

[0148] The membrane material was treated using a strong alkali and enhanced oxidant process. It underwent degreasing, protein removal, and DNA residue removal. The membrane material was then freeze-dried / dehydrated and self-assembled. The resulting material was observed and measured. The results are shown in Table 9.

[0149] Table 9

[0150]

[0151] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for self-assembling a natural collagen decellularized matrix scaffold material, characterized in that, The method includes the following steps: (1) Fresh animal membrane tissue was immersed in a pretreatment solution to remove cells, fat, impurities, proteins, and DNA residues, and the first membrane material was obtained. (2) The first membrane material is immersed in an ethanol solution and dried to obtain the second membrane material; (3) Immerse the second membrane material in the first immersion solution and dry it to obtain the third membrane material; (4) Immerse the third membrane material in the second immersion solution, dry it, and then proceed with the fourth membrane material process; (5) The fourth membrane material is cut, packaged, and sterilized to obtain the natural collagen decellularized matrix scaffold material; The first soaking solution / second soaking solution contains glycerol and glycine.

2. The self-assembly method of natural collagen decellularized matrix scaffold material according to claim 1, characterized in that, The first / second soaking solution comprises, by volume concentration: 2-20 g of glycerol per 100 ml, 0.1-0.5 g of glycine per 100 ml, and the remainder is a buffer solution with pH 5-8.

3. The self-assembly method of natural collagen decellularized matrix scaffold material according to claim 1, characterized in that, In step (1), the pretreatment solution comprises, by weight percentage: 0.5-10% alkali, 1-10% oxide, 0.5-5% degreasing agent, and the balance being water; wherein, The alkali is sodium hydroxide, potassium hydroxide, sodium carbonate, trisodium phosphate, sodium tripolyphosphate, or sodium pyrophosphate; the oxide is hydrogen peroxide; and the degreasing agent is Tween 80, Triton X-100, or fatty alcohol polyoxyethylene ether AEO-9. The soaking temperature is 0-25°C, and the soaking time is 20-60 hours.

4. The self-assembly method of natural collagen decellularized matrix scaffold material according to claim 1, characterized in that, In step (2), the concentration of the ethanol solution is 50-90%; the soaking temperature is 0-10℃ and the time is 1-5h; the drying temperature is 30-40℃ and the time is 0.5-5h.

5. The self-assembly method of natural collagen decellularized matrix scaffold material according to claim 1, characterized in that, In step (3), the soaking temperature is 0-10℃ and the time is 0.5-5h; the drying temperature is 30-40℃ and the time is 0.5-5h.

6. The self-assembly method of the natural collagen decellularized matrix scaffold material according to claim 1, characterized in that, In step (4), the soaking temperature is 10-25℃ and the time is 0.5-5h; the drying temperature is 30-55℃ and the time is 0.5-5h.

7. The self-assembly method of natural collagen decellularized matrix scaffold material according to claim 2, characterized in that, The concentration of the buffer solution is 0.05-0.5 mol / L, and the buffer solution is a phosphate buffer and / or a citrate buffer; the phosphate solution includes phosphoric acid, sodium phosphate, and potassium phosphate; the citrate solution includes citric acid, sodium citrate, and potassium citrate.

8. A natural collagen decellularized matrix scaffold material, characterized in that, It is made by the method of any one of claims 1-7.

Images