CN113663137B

CN113663137B - Composite biological patch and preparation method and application thereof

Info

Publication number: CN113663137B
Application number: CN202110964615.6A
Authority: CN
Inventors: 聂洪涛; 张凯; 王璇
Original assignee: Beijing Bonsci Technology Co ltd
Current assignee: Beijing Bonsci Technology Co ltd
Priority date: 2021-08-19
Filing date: 2021-08-19
Publication date: 2022-12-06
Anticipated expiration: 2041-08-19
Also published as: CN113663137A

Abstract

The invention provides a composite biological patch and a preparation method and application thereof, and relates to the technical field of medical treatment. The composite biological patch provided by the invention comprises a hydrophobic porous layer, a compact layer and a porous support, wherein the hydrophobic porous layer is back to one side of a tissue to be repaired and is loaded with anti-infective drugs, and the hydrophobicity of the hydrophobic porous layer and the loaded drugs can be matched with each other, so that the composite biological patch is beneficial to slow release of the drugs and plays roles of preventing and resisting infection and preventing adhesion; the porous scaffold has a porous structure, faces one side of the tissue to be repaired, is beneficial to the adhesion and growth of cells and induces the regeneration of membrane tissue; the hydrophobic porous layer and the porous support are separated by the compact layer, so that the dimensional stability and the mechanical strength of a product can be ensured, and meanwhile, the function of a barrier can be played. The composite biological patch provided by the invention can be used for meninges repair, rotator cuff repair, hernia repair, ophthalmologic repair, breast repair, hemostatic repair or oral repair.

Description

Composite biological patch and preparation method and application thereof

Technical Field

The invention relates to the technical field of medical treatment, in particular to a composite biological patch and a preparation method and application thereof.

Background

The repair and regeneration of human defective tissues are always difficult problems in clinic. In recent years, with the development of cell biology and tissue engineering technology, a new repair material, namely a biological patch, is receiving attention. The biological patch is a material which is derived from homologous or heterologous biological tissues, removes various cells contained in the tissues through treatment processes such as decellularization and the like to completely preserve the three-dimensional structure of extracellular matrix and can be used for repairing damaged soft tissues of a human body. The biological patch can repair damaged tissues through space induction and tissue replacement, and has good tissue compatibility. Collagen is an extracellular matrix, a structural protein with biological functions, accounts for 1/3 of the total amount of human proteins, and is a main component constituting connective tissues or organs such as skin, ligament, cartilage, tendon, and the like. As a biomedical material, collagen has excellent properties such as weak immunoantigenicity, good biodegradability, and promotion of cell survival and growth. Therefore, the collagen basement membrane patch is widely applied to the aspects of dural membrane defect repair, motor tendon laceration repair, hernia and abdominal wall defect repair, burn plastic, oral cavity membrane defect repair and the like.

At present, the main material sources of the collagen-based patch comprise an allogeneic material and a xenogeneic material, and the allogeneic material is not suitable for clinical application because the material sources are easily limited, protein viruses easily exist and the price is relatively expensive. The xenogenic materials comprise pigskin, pig small intestine, bovine pericardium, bovine achilles tendon and the like, and the natural materials are derived from tissues of pigs, cows and the like, so that the natural materials have the advantages of abundant resources, low cost, small toxicity after treatment and good biocompatibility, and therefore, the biological patch prepared from the raw materials has wide application prospect. However, the current bioprosthesis has the following disadvantages: (1) the general mechanical property is not ideal enough, in order to ensure the mechanical strength of the patch, a chemical crosslinking method is mostly adopted for modification, solvent residue is easy to occur, and the biocompatibility and the safety of the patch cannot be ensured; (3) animal tissues such as pigskin and pericardium are directly treated and then repaired, so that the plaster has poor adhesion and is easy to shift; (4) does not have a suitable pore structure and thus does not have good tissue regeneration properties.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

A first object of the present invention is to provide a composite biological patch to solve at least one of the above problems.

The second purpose of the invention is to provide a preparation method of the composite biological patch, which is simple and convenient and has low cost.

The third purpose of the invention is to provide the application of the composite biological patch in meninges repair, spinal cord repair, rotator cuff repair, hernia repair, ophthalmology repair, breast repair, hemostasis repair or oral repair.

In a first aspect, the present invention provides a composite biological patch comprising a hydrophobic porous layer, a dense layer and a porous scaffold;

the hydrophobic porous layer is back to one side of the tissue to be repaired and is loaded with anti-infective drugs;

the side of the porous bracket facing the tissue to be repaired;

the dense layer is located between the hydrophobic porous layer and the porous scaffold.

As a further technical scheme, the raw material for preparing the hydrophobic porous layer comprises hydrophobic nano-cellulose;

preferably, the hydrophobic nanocellulose comprises ethylcellulose.

As a further technical scheme, the preparation method of the hydrophobic porous layer comprises the following steps:

dissolving hydrophobic nano-cellulose in an organic reagent, adding an anti-infective drug, and preparing a membrane after mixing;

preferably, the organic reagent comprises hexafluoroisopropanol and/or ethanol;

preferably, the ratio of the hydrophobic nanocellulose to the organic reagent is 1g (4-8) mL;

preferably, the anti-infective drug is added in an amount of 5-10% of the hydrophobic nano-cellulose in percentage by mass;

preferably, the film-making manner comprises electrostatic spinning;

preferably, the thickness of the hydrophobic porous layer is 0.5-2mm.

As a further technical scheme, the preparation material of the compact layer comprises ethyl cellulose and/or hydroxypropyl methyl cellulose.

According to a further technical scheme, the preparation material of the porous scaffold comprises at least one of type I collagen, cellulose, chitosan, hyaluronic acid, gelatin, silk fibroin and starch, and preferably the type I collagen.

As a further technical scheme, the preparation method of the porous scaffold comprises the following steps:

dissolving the type I collagen in an acid solution to obtain a collagen solution, and then drying to prepare a porous scaffold;

preferably, the acidic solution comprises an acetic acid solution and/or a hydrochloric acid solution, preferably an acetic acid solution;

preferably, the concentration of the acetic acid solution is 0.01-1mol/L, preferably 0.02-0.5mol/L;

preferably, the concentration of the type I collagen in the collagen solution is 0.1wt% to 2.0wt%, preferably 0.5wt% to 1.5wt%;

preferably, the manner of drying comprises freeze drying;

preferably, the freeze-drying conditions are as follows: treating at-40 deg.c to-10 deg.c for 1-6 hr, treating at-10 deg.c to 0 deg.c for 12-48 hr, and treating at 22 deg.c to 28 deg.c for 1-2 hr.

As a further technical scheme, the preparation method of the type I collagen comprises the following steps:

a. removing impurities from the animal achilles tendon, crushing, and then carrying out salt solution treatment;

b. treating the animal achilles tendon treated in the step a with enzyme, and removing insoluble substances;

c. b, adjusting the pH value of the solution obtained in the step b to 12-14, and adding a salt solution until the salt concentration in the solution is 2.5-7.5mol/L to obtain a precipitate;

d. c, sequentially treating the precipitate obtained in the step c with an acidic solution, separating and drying to obtain type I collagen;

preferably, the animal achilles tendon comprises at least one of bovine achilles tendon, porcine achilles tendon, ma Genjian, and ovine achilles tendon, preferably bovine achilles tendon;

preferably, in step a, the salt solution comprises NaCl solution and NaHCO solution ₃ At least one of solution, sodium citrate solution, tris-HCl solution and Tris-Base solution, preferably NaCl solution;

preferably, in the step a, the concentration of the salt solution is 5wt% to 25wt%;

preferably, in the step a, the mass ratio of the animal achilles tendon to the saline solution is 1 (50-100);

preferably, in the step b, the enzyme treatment is to treat the animal achilles tendon with an acid solution containing enzyme;

preferably, in step b, the enzyme comprises at least one of pepsin, trypsin, ficin, bromelain, papain, griseofulvin and subtilisin, preferably pepsin;

preferably, in the step b, the acidic solution comprises at least one of an acetic acid solution, a citric acid solution, a malic acid solution and a lactic acid solution, and is preferably an acetic acid solution;

preferably, in the step b, the concentration of the acid solution is 0.25-0.8mol/L;

preferably, in step c, the pH of the solution obtained in step b is adjusted by using alkali solution, wherein the alkali comprises NaOH, KOH, ca (OH) ₂ Preferably NaOH;

preferably, in the step c, the pH value of the solution obtained in the step b is adjusted to 13, and a salt solution is added until the salt concentration in the solution is 4-5mol/L;

preferably, in the step d, the acidic solution includes at least one of a hydrochloric acid solution, an acetic acid solution, a citric acid solution, a phosphoric acid solution and a sulfuric acid solution, and is preferably a hydrochloric acid solution;

preferably, in the step d, the concentration of the acidic solution is 0.1-10mol/L, and preferably 2-5mol/L.

As a further technical scheme, the anti-infective drug comprises at least one of ketoprofen, roxithromycin, amoxicillin or ciprofloxacin.

In a second aspect, the invention provides a preparation method of a composite biological patch, which comprises the following steps:

mixing an organic reagent dissolved with ethyl cellulose and a hydroxypropyl methyl cellulose aqueous solution, then coating the mixed solution between the hydrophobic porous layer and the porous bracket, pressing, and heating to obtain a composite biological patch;

preferably, the organic reagent comprises ethanol;

preferably, the concentration of ethyl cellulose in the organic reagent is 2wt% to 5wt%;

preferably, the concentration of the hydroxypropyl methyl cellulose aqueous solution is 2wt% -5wt%;

preferably, the volume ratio of the organic reagent dissolved with the ethyl cellulose to the hydroxypropyl methyl cellulose aqueous solution is 1 (10-15);

preferably, the pressure of the pressing is 0.4-0.8MPa;

preferably, the pressing time is 40-60min;

preferably, the temperature of the heating treatment is 110-130 ℃;

preferably, the time of the heat treatment is 24-36h;

preferably, the thickness of the mixed solution coating is 0.1-0.5mm;

preferably, the thickness of the composite biological patch is 2-5mm.

In a third aspect, the invention provides a composite biological patch for meninges repair, spinal membrane repair, rotator cuff repair, hernia repair, ophthalmic repair, breast repair, hemostatic repair or oral repair.

Compared with the prior art, the invention has the following beneficial effects:

the composite biological patch provided by the invention comprises a hydrophobic porous layer, a compact layer and a porous support, wherein the hydrophobic porous layer is back to one side of a tissue to be repaired and is loaded with anti-infective drugs, and the hydrophobicity of the hydrophobic porous layer and the loaded drugs can be matched with each other, so that the composite biological patch is beneficial to slow release of the drugs and plays roles of preventing and resisting infection and preventing adhesion; the porous scaffold has a porous structure, faces one side of the tissue to be repaired, is beneficial to the adhesion and growth of cells and induces the regeneration of membrane tissue; the hydrophobic porous layer and the porous support are separated by the compact layer, so that the dimensional stability and the mechanical strength of a product can be ensured, and meanwhile, the function of a barrier can be played.

The preparation method of the composite biological patch provided by the invention is simple and convenient, and has low cost.

The composite biological patch provided by the invention can be used for meninges repair, rotator cuff repair, hernia repair, ophthalmologic repair, breast repair, hemostatic repair or oral repair.

Drawings

In order 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 described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a graph of the drug release rates of the membrane patches of examples 1-3 and comparative example 3;

FIG. 2 is a graph of the membrane patch staining of example 1;

figure 3 is a graph of the membrane patch staining of comparative example 3.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Those who do not specify the conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The composite biological patch can be used as a meninges patch, a rotator cuff patch, a hernia patch, an ophthalmological patch, a breast patch, a hemostatic patch or an oral cavity repairing film, wherein the side back to the tissue to be repaired in the invention refers to the side back to the meninges, the rotator cuff, the oral cavity film and other defective tissues; the side facing the tissue to be repaired is the side facing other defected tissues such as meninges, spinal cord membrane, rotator cuff, oral cavity membrane and the like.

In a first aspect, the present invention provides a composite biological patch comprising a hydrophobic porous layer, a dense layer, and a porous scaffold;

the side of the porous bracket facing the tissue to be repaired;

The composite biological patch provided by the invention comprises a hydrophobic porous layer, a compact layer and a porous support, wherein the hydrophobic porous layer is back to one side of a tissue to be repaired and is loaded with anti-infective drugs, and the hydrophobicity of the hydrophobic porous layer and the loaded drugs can be matched with each other, so that the composite biological patch is beneficial to slow release of the drugs and plays roles of preventing and resisting infection and preventing adhesion; the porous scaffold has a porous structure, faces one side of the tissue to be repaired, is beneficial to the adhesion and growth of cells and induces the regeneration of membranous tissue; the hydrophobic porous layer and the porous support are separated by the compact layer, so that the dimensional stability and the mechanical strength of a product can be ensured, and meanwhile, the function of a barrier can be played.

In some preferred embodiments, the raw material for preparing the hydrophobic porous layer includes, but is not limited to, hydrophobic nanocellulose, and the preparation of the hydrophobic porous layer using the hydrophobic nanocellulose as the raw material can significantly improve the mechanical properties of the hydrophobic porous layer.

Preferably, the hydrophobic nanocellulose includes, but is not limited to, ethylcellulose.

Ethyl Cellulose (EC) is a water-insoluble cellulose derivative in which hydroxyl groups on the molecule are substituted with a large number of ethyl groups, and unsubstituted hydroxyl groups are connected by hydrogen bonds to form a compact coral-like network structure, giving EC excellent mechanical properties, and its non-toxic, hydrophobic, mechanical, thermoplastic and film-forming properties are of value in many fields such as food, microencapsulation, filtration and medicine. In the invention, the hydrophobic nano-cellulose prepared by taking the ethyl cellulose as the raw material has good mechanical property and hydrophobicity, and can play a role in drug slow release.

In some preferred embodiments, the method of preparing the hydrophobic porous layer comprises the steps of:

preferably, the organic reagent includes, but is not limited to, hexafluoroisopropanol and/or ethanol;

preferably, the ratio of the hydrophobic nanocellulose to the organic reagent is 1g (4-8) mL, and may be, for example, but not limited to, 1g;

preferably, the anti-infective agent is added in an amount of 5% -10% by mass of the hydrophobic nanocellulose, such as but not limited to 5%, 6%, 7%, 8%, 9% or 10%;

preferably, the film-making method includes but is not limited to electrospinning, or other film-making methods known to those skilled in the art;

preferably, the thickness of the hydrophobic porous layer is 0.5-2mm, for example, but not limited to, 0.5mm, 1mm, 1.5mm or 2mm.

The preparation method of the hydrophobic porous layer is further optimized and adjusted, so that the hydrophobic porous layer has good mechanical properties, can slowly release medicines, and has the effects of preventing and resisting infection and adhesion.

In some preferred embodiments, the dense layer is made from materials including, but not limited to, ethyl cellulose and/or hydroxypropyl methyl cellulose. The hydroxypropyl methyl cellulose has the advantages of good film forming property, adhesion, oxidation resistance, broad-spectrum antibacterial property, excellent barrier property and the like, the compact layer prepared by taking the hydroxypropyl methyl cellulose as a raw material has good compactness, and meanwhile, the connection strength between the hydroxypropyl methyl cellulose and the hydrophobic porous layer and the porous support can be enhanced. The ethyl cellulose has hydrophobicity, and the connection strength between the dense layer and the hydrophobic porous layer can be improved by using the ethyl cellulose as a raw material to prepare the dense layer.

The term "and/or" means that the preparation material of the dense layer may include ethyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose and hydroxypropyl methyl cellulose.

In some preferred embodiments, the porous scaffold is made of a material including, but not limited to, at least one of type i collagen, cellulose, chitosan, hyaluronic acid, gelatin, silk fibroin, and starch, preferably type i collagen.

The porous scaffold prepared by taking the type I collagen as a raw material has a three-dimensional porous structure, is more favorable for cell adhesion and growth, and induces regeneration of membranous tissues. It should be noted that the source of the type I collagen in the present invention is not particularly limited, and commercially available type I collagen or self-prepared type I collagen may be used.

In some preferred embodiments, the method for preparing the porous scaffold comprises the steps of:

dissolving the I type collagen in an acid solution to obtain a collagen solution, and then carrying out drying treatment to prepare a porous scaffold;

preferably, the acidic solution includes, but is not limited to, an acetic acid solution and/or a hydrochloric acid solution, preferably an acetic acid solution;

preferably, the concentration of the acetic acid solution is 0.01-1mol/L, such as but not limited to 0.01mol/L, 0.05mol/L, 0.1mol/L, 0.2mol/L, 0.4mol/L, 0.6mol/L, 0.8mol/L or 1mol/L, preferably 0.02-0.5mol/L;

preferably, the concentration of type i collagen in the collagen solution is between 0.1wt% and 2.0wt%, such as but not limited to 0.1wt%, 0.2wt%, 0.5wt%, 1.0wt%, 1.5wt% or 2.0wt%, preferably between 0.5wt% and 1.5wt%;

preferably, the drying means includes, but is not limited to, freeze drying, or other drying means known to those skilled in the art;

preferably, the freeze-drying conditions are as follows: treating at-40 deg.c to-10 deg.c for 1-6 hr, treating at-10 deg.c to-0 deg.c for 12-48 hr, and treating at 22 deg.c to 28 deg.c for 1-2 hr.

Preferably, the freeze-drying process conditions are: treating at-30 deg.c to-20 deg.c for 1-6 hr, treating at-10 deg.c to 0 deg.c for 24-36 hr, and treating at 22 deg.c to 28 deg.c for 1-2 hr.

In the invention, the preparation method of the porous scaffold is further optimized and adjusted, so that the prepared porous scaffold has a good three-dimensional porous structure, and is more favorable for cell adhesion and growth and membrane tissue regeneration induction.

In some preferred embodiments, the method for preparing the type i collagen comprises the steps of:

a. removing impurities from the Achilles tendon of the animal, crushing, and then carrying out salt solution treatment. Wherein the impurity removal is to remove excessive impurities such as tissues, fat and the like; the salt solution is treated to remove contaminating proteins. One of the most suitable raw materials for preparing type I collagen is Achilles tendon, and the fibers of the Achilles tendon are almost all type I collagen, so that the steps required for purifying the type I collagen are greatly simplified. Therefore, the invention prepares the type I collagen by using the animal achilles tendon raw material.

b. And (b) performing enzyme treatment on the animal achilles tendon treated in the step (a), and removing insoluble substances. Wherein the enzyme treatment is to remove other impurity proteins; the insoluble matter is mainly large-sized Achilles tendon tissue, and needs to be removed, or the large-sized Achilles tendon can be pulverized again and then treated with enzyme again to improve the utilization rate of the raw material.

c. And c, adjusting the pH value of the solution obtained in the step b to 12-14, and adding a salt solution until the salt concentration in the solution is 2.5-7.5mol/L to obtain a precipitate. The isoelectric precipitation method and the salting-out method are adopted to separate I type collagen precipitate, in addition, the alkaline condition can inactivate the virus well, and the biocompatibility and the safety of the collagen can be improved.

d. And c, sequentially treating the precipitate obtained in the step c with an acidic solution, separating and drying to obtain the type I collagen. And dissolving the precipitate again to remove insoluble impurities, thereby further improving the purity of the type I collagen.

Preferably, the animal achilles tendon comprises but is not limited to at least one of bovine achilles tendon, porcine achilles tendon, ma Genjian, ovine achilles tendon, preferably bovine achilles tendon;

preferably, in step a, the salt solution includes, but is not limited to, naCl solution, naHCO solution ₃ At least one of solution, sodium citrate solution, tris-HCl solution and Tris-Base solution, preferably NaCl solution;

preferably, in step a, the concentration of the salt solution is 5wt% to 25wt%, for example, but not limited to, 5%, 10%, 15%, 20% or 25%;

preferably, in the step a, the mass ratio of the animal achilles tendon to the saline solution is 1 (50-100), and can be, but is not limited to, 1;

preferably, in step b, the enzyme includes but is not limited to at least one of pepsin, trypsin, ficin, bromelain, papain, streptomyces griseus secretase and subtilase, preferably pepsin;

preferably, in step b, the acidic solution includes but is not limited to at least one of acetic acid solution, citric acid solution, malic acid solution and lactic acid solution, preferably acetic acid solution;

preferably, in step b, the concentration of the acidic solution is 0.25-0.8mol/L, such as but not limited to 0.25mol/L, 0.3mol/L, 0.4mol/L, 0.5mol/L, 0.6mol/L, 0.7mol/L or 0.8mol/L;

preferably, in step c, the pH of the solution obtained in step b is adjusted with a base solution, including but not limited to NaOH, KOH, ca (OH) ₂ Preferably NaOH;

preferably, in the step c, the pH value of the solution obtained in the step b is adjusted to 13, and a salt solution is added to the solution to make the salt concentration in the solution be 4-5mol/L, under the condition, the collagen separation effect is best.

Preferably, in step d, the acidic solution includes but is not limited to at least one of hydrochloric acid solution, acetic acid solution, citric acid solution, phosphoric acid solution and sulfuric acid solution, preferably hydrochloric acid solution;

preferably, in step d, the concentration of the acidic solution is 0.1-10mol/L, for example, but not limited to, 0.1mol/L, 0.2mol/L, 0.5mol/L, 1mol/L, 2mol/L, 4mol/L, 6mol/L, 8mol/L or 10mol/L, preferably 2-5mol/L.

The preparation method of the I-type collagen has the following advantages:

1. the animal achilles tendon is taken as a raw material to extract the collagen, the treatment process is simple, and the purity of the collagen is high;

2. the purity of the extracted collagen is high and reaches more than 99 percent, and the triple-helix structure of the collagen is well maintained, so that the collagen has low immunogenicity;

3. the high-purity collagen is the basis and guarantee of the excellent tissue regeneration performance of the product;

4. the virus is well inactivated by an alkali treatment process, so that the extracted collagen has good biocompatibility and safety.

In some preferred embodiments, the anti-infective drug includes, but is not limited to, at least one of ketoprofen, roxithromycin, amoxicillin, or ciprofloxacin, or other anti-infective drugs known to those of skill in the art.

preferably, the organic agent includes, but is not limited to, ethanol, or other organic agents known to those skilled in the art;

preferably, the concentration of ethylcellulose in the organic reagent is from 2wt% to 5wt%, for example, but not limited to, 2wt%, 3wt%, 4wt%, or 5wt%;

preferably, the concentration of the hydroxypropyl methylcellulose aqueous solution is 2wt% to 5wt%, for example, may be, but is not limited to, 2wt%, 3wt%, 4wt%, or 5wt%;

preferably, the volume ratio of the organic reagent dissolved with ethyl cellulose to the hydroxypropyl methylcellulose aqueous solution is 1 (10-15), and can be, but is not limited to 1;

preferably, the pressure of the pressing is 0.4-0.8MPa, such as but not limited to 0.4MPa, 0.5MPa, 0.6MPa, 0.7MPa or 0.8MPa;

preferably, the pressing time is 40-60min, such as but not limited to 40min, 45min, 50min, 55min or 60min;

preferably, the temperature of the heat treatment is 110 to 130 ℃, for example, but not limited to, 110 ℃, 115 ℃, 120 ℃, 125 ℃ or 130 ℃;

preferably, the time of the heat treatment is 24-36h, for example, but not limited to 24h, 26h, 28h, 30h, 32h, 34h or 36h;

preferably, the mixed solution is coated to a thickness of 0.1 to 0.5mm, such as but not limited to 0.1mm, 0.2mm, 0.3mm, 0.4mm, 0.5mm, or 0.6mm;

preferably, the composite biological patch has a thickness of 2-5mm, such as but not limited to 2mm, 3mm, 4mm, 5mm or 6mm.

In the invention, the preparation method of the composite biological patch is further optimized and adjusted, so that the compactness of the dense layer and the connection strength between the dense layer and the hydrophobic porous layer as well as the porous support are further improved.

The composite biological patch has excellent mechanical property and high flexibility, is easy to be attached to a damaged part, can avoid suturing, and can effectively prevent adhesion and induce tissue regeneration; the middle connecting layer and the other two layers have good connecting strength and good compactness, can prevent tissue fluid from leaking and play a role of a barrier; the support layer has good hydrophilicity, the hydrophobic porous layer has good hydrophobicity, and the anti-infection and anti-adhesion effects are good. Can be used as a meningeal patch, rotator cuff patch, hernia patch, ophthalmic patch, breast patch, hemostatic patch, and oral cavity restoration membrane.

The invention is further illustrated by the following specific examples and comparative examples, but it should be understood that these examples are for purposes of illustration only and are not to be construed as limiting the invention in any way.

Example 1

A composite biological patch is prepared by the following steps:

1. preparation of the hydrophobic porous layer: dissolving a certain amount of ethyl cellulose in hexafluoroisopropanol solvent, adding 1g of the ethyl cellulose into the hexafluoroisopropanol solvent at a ratio of 6mL, stirring for 36h to prepare a spinning solution with a concentration of 9.5%, then adding the anti-infective drug ketoprofen, adding the ethyl cellulose at a ratio of 8%, and then continuously stirring for 60h to obtain the spinning solution containing the anti-infective drug. And (2) putting the spinning solution into an injector, connecting a needle head with high pressure, adopting an aluminum foil as a receiving device, setting the spinning voltage at 15kV, the spinning speed at 0.3mL/h, the receiving distance at 15cm and the spinning time at 3h to obtain a cellulose nanofiber membrane with the thickness of 1mm, then placing the obtained nanofiber membrane in a vacuum oven, drying at 80 ℃ overnight, and removing the residual solvent.

2. Preparation of type I collagen porous scaffold:

preparation of type I collagen: weighing a certain weight of fresh bovine achilles tendon material, pretreating, sufficiently removing redundant tissues, fat and other impurities, sufficiently crushing the treated bovine achilles tendon, soaking in a 15% sodium chloride solution (the mass ratio of the bovine achilles tendon to the salt solution is 1: 75) for 6 hours, washing with sterilized distilled water for several times, and centrifuging to obtain the pretreated bovine achilles tendon;

suspending the treated Achilles tendon in acetic acid solution (acetic acid concentration is 0.5 mol/L) of enzyme for extraction, and fully extracting collagen by adopting special stirring and cooling equipment in the extraction process and maintaining the temperature at a lower value. Centrifuging after stirring and extracting for 1h to remove the precipitate, preserving the supernatant, then crushing the removed precipitate again and stirring for 1h, centrifuging again and removing the precipitate, continuously repeating the process to carry out re-extraction, and obtaining the supernatant again and preserving;

adjusting the pH of the obtained supernatant to be final pH =13 by using a sodium hydroxide solution, then adding a NaCl solution until the concentration of NaCl in the solution is 4mol/L, stirring for 33h, and centrifuging to obtain a precipitate, namely a high-purity collagen semi-finished product;

and (3) putting the obtained semi-finished product into a 3mol/L acetic acid solution for neutralization for 24 hours, and then filtering and drying the semi-finished product to obtain a high-purity type I collagen finished product (the purity is over 99 percent).

Preparation of collagen solution: dissolving high-purity I-type collagen in 0.6mol/L acetic acid solution, and strongly stirring for 24h to obtain 1% uniform collagen solution;

freeze-drying of collagen solution: the collagen solution obtained was freeze-dried.

And (3) a freeze drying process: firstly, putting a collagen solution into a freezing chamber (the temperature is-30 ℃ to-20 ℃) for freezing for 3 hours, then raising the temperature of the freezing chamber to keep the temperature between-10 ℃ and 0 ℃ for 30 hours, after the collagen solution is frozen and dried to a certain degree, gradually keeping the temperature in the freezing and drying chamber to the room temperature for 1.5 hours, and taking out the collagen solution to obtain the collagen scaffold material with the bionic three-dimensional porous structure.

3. Preparation of a film patch: preparation of a connecting layer solution: weighing a certain amount of ethyl cellulose, dissolving the ethyl cellulose in an absolute ethyl alcohol solution, wherein the solution concentration is 3%, simultaneously weighing a certain amount of hydroxypropyl methyl cellulose, dissolving the hydroxypropyl methyl cellulose in water, wherein the solution concentration is 3%, then mixing the ethyl cellulose solution and the hydroxypropyl methyl cellulose solution (the mass ratio of the ethyl cellulose solution to the hydroxypropyl methyl cellulose solution is 1.

Example 2

A composite biological patch is prepared by the following steps:

1. preparation of the hydrophobic porous layer: dissolving a certain amount of ethyl cellulose in absolute ethyl alcohol serving as a solvent, wherein the addition ratio is 1g, namely 4mL, stirring for 24-48h to prepare a spinning solution with the concentration of 24.1%, then adding the anti-infective drug ketoprofen, wherein the addition ratio is 5% of the ethyl cellulose, and then continuously stirring for 48h to obtain the spinning solution containing the anti-infective drug. And (2) putting the spinning solution into an injector, connecting a needle head with high pressure, adopting an aluminum foil as a receiving device, setting the spinning voltage to 10kV, setting the spinning speed to 0.1mL/h, setting the receiving distance to 13cm and the spinning time to 1h to obtain a cellulose nanofiber membrane with the thickness of 0.5mm, then placing the obtained nanofiber membrane in a vacuum oven to dry overnight at 80 ℃, and removing the residual solvent.

2. Preparation of type I collagen porous scaffold:

type I collagen was prepared as in example 1.

Preparation of collagen solution: dissolving high-purity I type collagen in 0.025mol/L hydrochloric acid solution, and strongly stirring for 24h to obtain uniform collagen solution with the concentration of 0.1%;

And (3) a freeze drying process: firstly, putting a collagen solution into a freezing chamber (the temperature is-40 ℃ to-20 ℃) for freezing for 1h, then raising the temperature of the freezing chamber to keep the temperature between-10 ℃ and 0 ℃, keeping the time for 12h, preferably 24h, after the collagen solution is freeze-dried to a certain degree, gradually making the temperature in the freeze-drying chamber reach the room temperature, keeping the room temperature for 1h, and taking out the collagen solution to obtain the collagen scaffold material with the bionic three-dimensional porous structure.

3. Preparation of the film patch: preparation of the tie layer solution: weighing a certain amount of ethyl cellulose, dissolving the ethyl cellulose in an absolute ethyl alcohol solution, wherein the solution concentration is 2%, simultaneously weighing a certain amount of hydroxypropyl methyl cellulose, dissolving the hydroxypropyl methyl cellulose in water, wherein the solution concentration is 2%, then mixing the ethyl cellulose solution and the hydroxypropyl methyl cellulose solution (the mass ratio of the ethyl cellulose solution to the hydroxypropyl methyl cellulose solution is 1.

Example 3

A composite biological patch is prepared by the following steps:

1. preparation of the hydrophobic porous layer: dissolving a certain amount of ethyl cellulose in a hexafluoroisopropanol solvent as a solvent, wherein the addition ratio is 1g, 8mL, stirring for 48h to prepare a spinning solution with the concentration of 7.3%, then adding the anti-infective drug ketoprofen, the addition ratio is 10% of the ethyl cellulose, and then continuously stirring for 72h to obtain the spinning solution containing the anti-infective drug. And (2) putting the spinning solution into an injector, connecting a needle head with high pressure, setting a receiving device to be an aluminum foil, setting the spinning voltage to be 20kV, the spinning speed to be 0.5mL/h, the receiving distance to be 20cm and the spinning time to be 4h, obtaining a cellulose nano-fiber membrane with the thickness of 2mm, then placing the obtained nano-fiber membrane in a vacuum oven, drying at 80 ℃ overnight, and removing the residual solvent.

2. Preparation of type I collagen porous scaffold:

type I collagen was prepared as in example 1.

Preparation of collagen solution: dissolving high-purity I-type collagen in 1mol/L acetic acid solution, strongly stirring for 24h to obtain uniform collagen solution with the concentration of 2.0%, and then vacuumizing the collagen solution to fully remove bubbles in the solution;

freeze-drying of the collagen solution: the collagen solution obtained was freeze-dried.

And (3) a freeze drying process: firstly, putting a collagen solution into a freezing chamber (the temperature is-20 ℃ to-10 ℃) for freezing for 6 hours, then raising the temperature of the freezing chamber to keep the temperature between-10 ℃ and 0 ℃, keeping the time for 48 hours, preferably 36 hours, after the collagen solution is freeze-dried to a certain degree, gradually keeping the temperature in the freeze-drying chamber to the room temperature, keeping the temperature for 2 hours, and taking out the collagen solution to obtain the collagen scaffold material with the bionic three-dimensional porous structure.

3. Preparation of the film patch: preparation of a connecting layer solution: weighing a certain amount of ethyl cellulose, dissolving the ethyl cellulose in an absolute ethyl alcohol solution, wherein the solution concentration is 5%, simultaneously weighing a certain amount of hydroxypropyl methyl cellulose, dissolving the hydroxypropyl methyl cellulose in water, wherein the solution concentration is 5%, then mixing the ethyl cellulose solution and the hydroxypropyl methyl cellulose solution (the mass ratio of the ethyl cellulose solution to the hydroxypropyl methyl cellulose solution is 1.

Comparative example 1

A biological patch is different from example 1 in that only a type I collagen porous scaffold is prepared, and the prepared porous scaffold is pressed for 50min at a pressure of 0.6MPa and then placed in a vacuum oven for heat treatment (temperature: 120 ℃ C., time is 30 h) to obtain a membrane patch.

Comparative example 2

A composite biological patch differing from example 1 in that the hydrophobic porous layer is not loaded with an anti-inflammatory drug.

Comparative example 3

A composite biological patch, which is different from the composite biological patch in example 1 in that a hydrophobic porous layer is replaced by a hydrophilic nanocellulose porous layer, and the hydrophilic nanocellulose porous layer is prepared by the following method:

dissolving a certain amount of cellulose acetate in a mixed solvent of acetone and N, N-dimethylacetamide (the volume ratio of the acetone to the N, N-dimethylacetamide is 1:1), adding the solution at a ratio of 1g to 8mL, stirring for 36h to prepare a spinning solution with a concentration of 12.6%, adding the anti-infective drug ketoprofen, adding the ethyl cellulose at a ratio of 8%, and then continuously stirring for 60h to obtain the spinning solution containing the anti-infective drug. And (2) putting the spinning solution into an injector, connecting a needle head with high pressure, setting a receiving device to be an aluminum foil, setting the spinning voltage to be 15kV, setting the spinning speed to be 0.3mL/h, setting the receiving distance to be 15cm, setting the spinning time to be 3h, obtaining a cellulose nanofiber membrane with the thickness of 1mm, then placing the obtained nanofiber membrane in a vacuum oven to dry overnight at the temperature of 80 ℃, and removing residual solvent.

Test example 1

1. Drug release rate: 200mg of the membrane patches of examples 1, 2 and 3 and comparative example 3 were immersed in 50mL of PBS buffer solution with pH value of 7, placed on a shaker, 5mL of the solution was taken out at 1h, 5h, 24h, 48h, 72h, 120h, 168h and 336h, respectively, while the corresponding volume of PBS buffer solution was supplemented, and then the absorbance of the taken-out solution at the corresponding UV wavelength of the drug was measured using a UV-visible spectrophotometer. Then preparing drug solutions with different concentrations, carrying out absorbance test under corresponding concentrations, drawing a drug concentration-absorbance standard curve according to test results, and calculating the in-vitro cumulative release rate of the drug according to the standard curve, wherein the test results are shown in figure 1.

According to the test result of the accumulative release rate, the anti-infective medicament is in the hydrophobic porous layer, so that the medicament in the membrane patch of the embodiment is slowly released, the accumulative release rate reaches the highest and reaches the balance basically at the 7 th day, and the highest accumulative release rate can reach about 60 percent; in contrast, in the membrane patch of comparative example 3, the porous layer is hydrophilic, so the initial release rate is faster, the highest release rate is reached and balanced within 48h, and the cumulative release rate reaches about 80%, thus the hydrophobicity of the porous layer is favorable for the slow release of the drug, so the aim of continuously resisting infection is achieved.

2. Mechanical properties: the samples were cut into 20mm wide bars for tensile testing according to the method in GBT3923.1-2013, and the test results are shown in the following table.

	Tensile Strength (MPa)	Elongation at Break (%)
			Example 1	5.35±1.21	18.35±2.33
Example 2	4.76±2.01	15.67±2.51
			Example 3	5.93±1.88	22.17±1.89
Comparative example 1	1.02±0.58	7.23±1.93
			Comparative example 2	5.23±2.10	17.79±2.45
Comparative example 3	5.12±2.69	17.98±3.01

According to test results, the preparation of the intermediate dense layer and the hydrophobic porous layer has a large influence on the mechanical property of the membrane patch, the mechanical property of the membrane patch can be obviously improved along with the introduction of the dense layer and the hydrophobic porous layer, and the larger the proportion of the hydroxypropyl cellulose solution in the intermediate dense layer is, the higher the tensile strength of the membrane patch is, so that the prepared membrane patch has excellent mechanical property compared with a common collagen scaffold.

3. Tissue regeneration induction and adhesion prevention:

a new Zealand white rabbit is used as a model, a wound with the length of 4cm is cut at a dura mater defect part, then membrane patches provided in example 1 and comparative example 3 are implanted, corresponding tissue specimens are taken at 6 months for HE staining, and tissue regeneration and adhesion conditions are observed, wherein the results of example 1 and comparative example 3 are shown in a graph in fig. 2 and a graph in fig. 3 respectively. Wherein A is brain tissue and the arrow indicates a patch of membrane.

As can be seen from the pictures, after 6 months, the patch implanted in example 1 found that new dural tissue had grown instead of the original implant, and the dural defect site had no significant adhesion to the brain tissue and no significant inflammatory response. The patch implanted in comparative example 3 also has new dura mater tissue growth, but due to the good hydrophilicity of the porous layer, adhesion is easy to occur, so that the new tissue is tightly connected to the brain tissue, and other diseases such as tumor are easy to cause.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The composite biological patch is characterized by comprising a hydrophobic porous layer, a compact layer and a porous support;

the side of the porous bracket facing the tissue to be repaired;

the compact layer is positioned between the hydrophobic porous layer and the porous support;

the preparation material of the compact layer comprises ethyl cellulose and/or hydroxypropyl methyl cellulose;

the preparation raw material of the hydrophobic porous layer comprises hydrophobic nano-cellulose;

the preparation material of the porous scaffold is type I collagen.

2. The composite biological patch according to claim 1, wherein the hydrophobic nanocellulose comprises ethyl cellulose.

3. The composite biological patch as claimed in claim 1, wherein the preparation method of the hydrophobic porous layer comprises the steps of:

dissolving hydrophobic nano-cellulose in an organic reagent, then adding an anti-infective drug, and preparing a membrane after mixing.

4. The composite biological patch as claimed in claim 3, wherein the organic agent comprises hexafluoroisopropanol and/or ethanol;

the proportion of the hydrophobic nano-cellulose to the organic reagent is 1g (4-8) mL;

the addition amount of the anti-infective drug is 5 to 10 percent of the hydrophobic nano-cellulose according to the mass percentage;

the membrane preparation mode comprises electrostatic spinning;

the thickness of the hydrophobic porous layer is 0.5-2mm.

5. The composite biological patch according to claim 1, wherein the preparation method of the porous scaffold comprises the following steps:

and dissolving the I type collagen in an acid solution to obtain a collagen solution, and then drying to prepare the porous scaffold.

6. The composite biological patch according to claim 5, wherein the acidic solution comprises an acetic acid solution and/or a hydrochloric acid solution;

when the acid solution is an acetic acid solution, the concentration of the acetic acid solution is 0.01-1mol/L;

the concentration of the I type collagen in the collagen solution is 0.1wt% -2.0wt%;

the drying means includes freeze drying.

7. The composite biological patch according to claim 6, wherein the acidic solution is an acetic acid solution;

the concentration of the acetic acid solution is 0.02-0.5mol/L;

the concentration of the I type collagen in the collagen solution is 0.5wt% -1.5wt%;

the freeze drying treatment conditions are as follows: processing for 1-6h at-40 to-10 ℃, then processing for 12 to 48h at-10 to 0 ℃, and then processing for 1-2h at 22 to 28 ℃.

8. The composite biological patch according to claim 1, wherein the preparation method of the type i collagen comprises the following steps:

a. removing impurities from the Achilles tendon of the animal, crushing, and then carrying out salt solution treatment;

d. and c, sequentially treating the precipitate obtained in the step c with an acidic solution, separating and drying to obtain the type I collagen.

9. The composite biological patch as claimed in claim 8, wherein the animal achilles tendon comprises at least one of bovine achilles tendon, porcine achilles tendon, ma Genjian, ovine achilles tendon;

in the step a, the salt solution comprises NaCl solution and NaHCO solution ₃ At least one of a solution, a sodium citrate solution, a Tris-HCl solution and a Tris-Base solution;

in the step a, the concentration of the salt solution is 5-25 wt%;

in the step a, the mass ratio of the animal achilles tendon to the saline solution is 1 (50-100);

in the step b, the enzyme treatment is to treat the animal achilles tendon by adopting an acid solution containing enzyme;

in the step b, the enzyme comprises at least one of pepsin, trypsin, ficin, bromelin, papain, griseofulvin and subtilisin;

in the step b, the acid solution comprises at least one of an acetic acid solution, a citric acid solution, a malic acid solution and a lactic acid solution;

in the step b, the concentration of the acid solution is 0.25-0.8mol/L;

in the step c, the pH value of the solution obtained in the step b is adjusted by adopting an alkali solution, wherein the alkali comprises NaOH, KOH and Ca (OH) ₂ At least one of;

in the step c, adjusting the pH value of the solution obtained in the step b to 13, and adding a salt solution until the salt concentration in the solution is 4-5mol/L;

in the step d, the acid solution comprises at least one of a hydrochloric acid solution, an acetic acid solution, a citric acid solution, a phosphoric acid solution and a sulfuric acid solution;

in the step d, the concentration of the acid solution is 0.1-10mol/L.

10. The composite biological patch as claimed in claim 9, wherein the animal achilles tendon is bovine achilles tendon;

in the step a, the salt solution is a NaCl solution;

in the step b, the enzyme is pepsin;

in the step b, the acid solution is an acetic acid solution;

in the step c, adjusting the pH value of the solution obtained in the step b by adopting an alkali solution, wherein the alkali is NaOH;

in the step d, the acid solution is a hydrochloric acid solution;

in the step d, the concentration of the acid solution is 2-5mol/L.

11. The composite biological patch as claimed in claim 1, wherein the anti-infective drug comprises at least one of ketoprofen, roxithromycin, amoxicillin or ciprofloxacin.

12. The method for preparing a composite biological patch according to claim 1, wherein the method comprises the following steps:

and mixing an organic reagent dissolved with ethyl cellulose and a hydroxypropyl methyl cellulose aqueous solution, coating the mixed solution between the hydrophobic porous layer and the porous bracket, pressing, and heating to obtain the composite biological patch.

13. The method of making a composite biological patch as claimed in claim 12, wherein the organic agent comprises ethanol;

the concentration of the ethyl cellulose in the organic reagent is 2-5 wt%;

the concentration of the hydroxypropyl methyl cellulose aqueous solution is 2-5 wt%;

the volume ratio of the organic reagent dissolved with the ethyl cellulose to the hydroxypropyl methyl cellulose aqueous solution is 1 (10-15);

the pressure of the pressing is 0.4-0.8MPa;

the pressing time is 40-60min;

the temperature of the heating treatment is 110-130 ℃;

the time of the heating treatment is 24-36h;

the thickness of the mixed solution coating is 0.1-0.5mm;

the thickness of the composite biological patch is 2-5mm.

14. Use of a composite biological patch according to any one of claims 1-11 or a composite biological patch prepared by the preparation method of claim 12 or 13 in the preparation of meninges repair, spinal membrane repair, rotator cuff repair, hernia repair, ophthalmic repair, breast repair, haemostatic repair or oral repair products.