Detailed Description
The following detailed description of the present application will provide further details in order to make the above-mentioned objects, features and advantages of the present application more comprehensible. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. The present application may be embodied in many other forms than described herein and similarly modified by those skilled in the art without departing from the spirit of the application, whereby the application is not limited to the specific embodiments disclosed below.
In the present application, "first aspect", "second aspect", "third aspect", etc. are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor as implying an importance or quantity of the indicated technical features. Moreover, "first," "second," "third," etc. are for non-exhaustive list description purposes only, and it should be understood that no closed limitation on the number is made.
In the present application, "optional" means optional or not, that is, means any one selected from two parallel schemes of "with" or "without". If multiple "alternatives" occur in a technical solution, if no particular description exists and there is no contradiction or mutual constraint, then each "alternative" is independent.
In the present application, the numerical ranges are referred to as continuous, and include the minimum and maximum values of the ranges, and each value between the minimum and maximum values, unless otherwise specified. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range description features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to include any and all subranges subsumed therein.
In the application, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items. The term "plurality" in the present application means at least two, for example, two, three, etc., unless specifically defined otherwise.
The application provides an artificial biological membrane patch, which comprises a repair layer and an anti-adhesion layer arranged on one side of the repair layer, wherein the anti-adhesion layer is of a porous structure, the anti-adhesion layer comprises a first polysaccharide polymer with an average crosslinking degree of 50% -90% and a second polysaccharide polymer with an average crosslinking degree of 10% -30%, and the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the anti-adhesion layer is 1:1-4:1.
Post-operative adhesions refer to various abnormal tissue hyperplasia formed between an organ and adjacent organs or tissues after surgical trauma. As adhesions grow, tissue can take on different phenotypes, ranging from a thin fibrous membrane to a mixture of loose or dense fibrous tissue, nerves and blood vessels, and even scar tissue. Postoperative adhesions occur widely in various soft tissues such as the peritoneum, pericardium, uterus, tendons, dura mater, etc., often resulting in chronic pain, dysfunction of adjacent organs and some acute complications, severely reducing the quality of life of the patient, even endangering life. The adhesions in which the blood clot deposited within 1d-3d after surgery is gradually replaced by granulation tissue are "membranous adhesions". Proliferation of 3d-14d fibroblasts/myofibroblasts after surgery, the formation of vascularized tissue, known as "vascular adhesions". Thus reducing bleeding at an early stage, thereby reducing hematoma formation, and inhibiting fibroblast proliferation can be an effective way to alleviate adhesions.
The artificial biological membrane patch can provide mechanical support and early anti-adhesion barrier for brain tissue or spinal tissue, and after the artificial biological membrane patch is implanted into a wound site, a porous structure in an anti-adhesion layer of the artificial biological membrane patch interacts with platelets so as to adsorb the platelets and accelerate the coagulation process, form a gel isolation barrier layer, prevent early fibroblast infiltration and adhesion and avoid early adhesion. Then a large number of fibroblasts and new capillary networks enter the repair layer to form new collagen tissues, and gradually replace the collagen tissues in the implanted artificial biomembrane patch. The average crosslinking degree of the first polysaccharide polymer and the average crosslinking degree of the second polysaccharide polymer in the anti-adhesion layer are controlled, and the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer is in the range, so that the bonding force between the first polysaccharide polymer and the second polysaccharide polymer in the anti-adhesion layer and the repair layer is improved, the scar hyperplasia is reduced, and the anti-adhesion effect and the biosafety of the artificial biological membrane patch are effectively improved. It is understood that the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the anti-blocking layer includes, but is not limited to, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1.
In some embodiments, the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the anti-blocking layer is 2:1-4:1. The mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the anti-adhesion layer is controlled to be in the range, so that the bonding force between the first polysaccharide polymer and the second polysaccharide polymer in the anti-adhesion layer and the repair layer is further improved, scar hyperplasia is further reduced, and the anti-adhesion effect of the artificial biological membrane patch is further improved.
In some embodiments, the first polysaccharide polymer and the second polysaccharide polymer each independently comprise a biodegradable polysaccharide polymer.
In some embodiments, the biodegradable polysaccharide polymer comprises at least one of chitosan, chitosan derivatives, cellulose, and cellulose derivatives.
In some embodiments, the chitosan derivative comprises at least one of carboxymethyl chitosan, alkylated chitosan, quaternized chitosan, and acylated chitosan.
In some embodiments, the cellulose derivative comprises at least one of methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, and hydroxypropylmethyl cellulose.
In some embodiments, the material of the repair layer comprises an extracellular matrix.
In some embodiments, the average degree of cross-linking of the first polysaccharide polymer is 60% -90%. The average crosslinking degree of the first polysaccharide polymer is controlled to be in the range, so that the bonding force between the first polysaccharide polymer in the anti-adhesion layer and the repair layer is further improved, scar hyperplasia is further reduced, and the anti-adhesion effect of the artificial biological membrane patch is further improved.
In some embodiments, the average degree of cross-linking of the second polysaccharide polymer is 15% -30%. The average crosslinking degree of the second polysaccharide polymer is controlled to be in the range, so that the bonding force between the second polysaccharide polymer in the anti-adhesion layer and the repair layer is further improved, scar hyperplasia is further reduced, and the anti-adhesion effect of the artificial biological membrane patch is further improved.
In some embodiments, at least a portion of the first polysaccharide polymer and/or at least a portion of the second polysaccharide polymer in the anti-blocking layer is attached to the repair layer by at least one of electrostatic interactions and secondary bonds. Therefore, the bonding force between the first polysaccharide polymer and/or the second polysaccharide polymer in the anti-bonding layer and the repairing layer is further improved, and the anti-bonding effect of the artificial biological membrane patch is further improved. The secondary bonds include, but are not limited to, hydrogen bonds, van der Waals forces.
In some embodiments, the anti-blocking layer has a porosity of 50% -90%.
In some embodiments, the mass of the anti-blocking layer per unit area of the repair layer is 1mg/cm2~6mg/cm2, alternatively 2mg/cm2~4mg/cm2.
Another embodiment of the present application provides a method for preparing an artificial biomembrane patch, comprising the steps of:
Mixing a solution containing a first polysaccharide polymer and a solution containing a second polysaccharide polymer, and carrying out homogenization treatment to obtain a film forming liquid, wherein the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the film forming liquid is 1:1-4:1, the average crosslinking degree of the first polysaccharide polymer is 50% -90%, and the average crosslinking degree of the second polysaccharide polymer is 10% -30%;
setting a film forming liquid on one side of the material of the repair layer to obtain an intermediate;
and removing the solvent in the intermediate to form a repairing layer and an anti-adhesion layer with a porous structure, thereby obtaining the artificial biomembrane patch.
In the preparation method, the average crosslinking degree of the first polysaccharide polymer and the average crosslinking degree of the second polysaccharide polymer in the anti-adhesion layer and the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer are controlled within the ranges, so that the bonding force between the first polysaccharide polymer and the second polysaccharide polymer in the anti-adhesion layer and the repair layer is improved, scar hyperplasia is reduced, and the anti-adhesion effect and biosafety of the artificial biological film patch are effectively improved.
In some embodiments, the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the film-forming liquid is 2:1-4:1. Therefore, the bonding force between the first polysaccharide polymer and the second polysaccharide polymer in the anti-bonding layer and the repairing layer is further improved, scar hyperplasia is further reduced, and the anti-bonding effect of the artificial biological membrane patch is further improved.
In some embodiments, the ratio of the mass of the film-forming liquid to the area of the material contacting the repair layer of the film-forming liquid is 2g:10cm2~3.2g:10cm2.
In some embodiments, a method of preparing a solution comprising a first polysaccharide polymer comprises the steps of:
Mixing a first crosslinking monomer with a first solvent, and performing first emulsification treatment to obtain a first solution;
Mixing the first solution with a first crosslinking agent, and performing crosslinking reaction to obtain first gel;
And sequentially carrying out ion replacement on the first gel in buffer solution and water to obtain a solution containing the first polysaccharide polymer. Alternatively, the process conditions for the crosslinking reaction include a crosslinking time of 6 hours or more.
In the above embodiment, the first emulsifying treatment may promote the first crosslinking monomer in the first solution to sufficiently react with the first crosslinking agent, and the first gel is ion-exchanged with the buffer solution and water, so as to facilitate removal of the first crosslinking agent remaining in the solution containing the first polysaccharide polymer.
In some embodiments, the first crosslinking monomer in the first solution is 2% -4% by mass. It is understood that the above mass percentages include, but are not limited to, 2%, 2.2%, 2.4%, 2.6%, 2.8%, 3%, 3.2%, 3.4%, 3.6%, 3.8%, 4%.
In some embodiments, the first crosslinking monomer comprises a polysaccharide polymer A having a viscosity of 20 mPas to 60 mPas. It is understood that the above viscosity includes, but is not limited to, 20 mPas, 30 mPas, 40 mPas, 50 mPas, 60 mPas.
In some embodiments, the mass ratio of the first cross-linking agent to the first solution is 1:1000 to 3:1000, optionally 1:1000 to 2:1000. Therefore, the bonding force between the first polysaccharide polymer in the anti-adhesion layer and the repair layer is further improved, scar hyperplasia is further reduced, and the anti-adhesion effect of the artificial biological membrane patch is further improved. It is understood that the mass ratio of the first crosslinker to the first solution includes, but is not limited to, 1:1000, 1.5:1000, 2:1000, 2.5:1000, 3:1000.
In some embodiments, the mass ratio of the first gel to the buffer is 1:10 to 1:40.
In some embodiments, the mass ratio of the first gel to the water is 1:10 to 1:40.
In some embodiments, the first crosslinker comprises at least one of 1, 4-butanediol diglycidyl ether and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride.
In some embodiments, the buffer comprises at least one of a sodium phosphate buffer and a potassium phosphate buffer.
In some embodiments, the first solvent comprises water.
In some embodiments, the method of preparing a solution comprising a second polysaccharide polymer comprises the steps of:
mixing a second crosslinking monomer with a second solvent, and performing second emulsification treatment to obtain a second solution;
And mixing the second solution with a second crosslinking agent to carry out crosslinking reaction to obtain a solution containing the second polysaccharide polymer. It is understood that the process conditions of the second emulsification treatment may be the same as or different from the process conditions of the first emulsification treatment. The second crosslinking monomer reacts with the second crosslinking agent to produce a second polysaccharide polymer.
In some embodiments, the mass percent of the second crosslinking monomer in the second solution is 2% -4%. It is understood that the above mass percentages include, but are not limited to, 2%, 2.2%, 2.4%, 2.6%, 2.8%, 3%, 3.2%, 3.4%, 3.6%, 3.8%, 4%.
In some embodiments, the second crosslinking monomer comprises a polysaccharide polymer B having a viscosity of 20 mPas to 60 mPas. It is understood that the above viscosity includes, but is not limited to, 20 mPas, 30 mPas, 40 mPas, 50 mPas, 60 mPas.
In some embodiments, polysaccharide polymer a and polysaccharide polymer B each independently comprise at least one of chitosan, a chitosan derivative, cellulose, and a cellulose derivative. Optionally, the chitosan derivative comprises at least one of carboxymethyl chitosan, alkylated chitosan, quaternized chitosan, and acylated chitosan. Optionally, the cellulose derivative comprises at least one of methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, and hydroxypropylmethyl cellulose.
In some embodiments, the mass ratio of the second crosslinking agent to the second solution is 0.1:1000 to 0.4:1000, optionally 0.1:1000 to 0.2:1000. Therefore, the bonding force between the second polysaccharide polymer in the anti-adhesion layer and the repair layer is further improved, scar hyperplasia is further reduced, and the anti-adhesion effect of the artificial biological membrane patch is further improved. It is understood that the mass ratio of the second crosslinker to the second solution includes, but is not limited to, 0.1:1000, 0.15:1000, 0.2:1000, 0.25:1000, 0.3:1000, 0.35:1000, 0.4:1000.
In some embodiments, the second crosslinker comprises at least one of 1, 4-butanediol diglycidyl ether and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride.
In some embodiments, the second solvent comprises water.
The artificial biomembrane patch in the embodiment of the application can be obtained by regulating and controlling the process conditions of the preparation method.
In a further embodiment, the present application provides the use of the artificial biomembrane patch of the present application or the artificial biomembrane patch prepared by the above preparation method of the present application for preparing a repair product for dura mater or dura mater.
In order to further illustrate the present application, the following describes the technical scheme of the present application in detail with reference to specific embodiments. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
The artificial biomembrane patch was prepared as follows:
(1) Preparation of extracellular matrix (material of repair layer)
Removing redundant fat and accessory tissues in animal membrane tissues, repeatedly washing the animal membrane tissues with purified water or normal saline until blood color is removed, extracting the animal membrane tissues with an organic reagent according to a ratio of 1:6 (w/v), at room temperature and 1200rpm, oscillating and degreasing for 4 hours, replacing the organic reagent, continuing oscillating and degreasing for 16 hours, discarding the organic reagent, adding the purified water according to a ratio of 1:20 (w/v), at room temperature and 1200rpm, oscillating and cleaning for 10 times and 10 minutes/time, and obtaining degreased animal membrane tissues;
(1.2) adding an alkali solution to the defatted animal membrane tissue at a feed solution ratio of 1:10 (w/v), oscillating at room temperature and 120rpm for 60min, washing, and repeating for three times to obtain extracellular matrix (material of repair layer).
(2) Preparation of a solution comprising a first polysaccharide polymer and a second polysaccharide polymer
(2.1) Preparation of a solution comprising a first polysaccharide Polymer
(2.1.1) Weighing a certain amount of carboxymethyl chitosan (first crosslinking monomer) with viscosity of 37.2 mPas, preparing a solution with mass concentration of 2% by using water for injection (first solvent), stirring by using an emulsifying machine, placing the solution in 2-8 ℃ to swell for more than 15 hours, stirring again after the swelling is finished, and stirring for about 30 minutes to obtain a first solution;
(2.1.2) adding a first crosslinking agent 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) into the first solution, standing and crosslinking for more than 6 hours to form a first gel, wherein the mass ratio of the first crosslinking agent to the first solution is 1:1000;
(2.1.3) preparing PBS buffer solution, placing the first gel into the PBS buffer solution for standing according to the mass ratio of the first gel to the PBS buffer solution of 1:20, and performing ion exchange by utilizing an osmotic pressure principle, wherein the solution is exchanged for 7h;
(2.1.4) repeating the step (2.1.3), wherein the replacement liquid is water for injection, and the mass ratio of the water for injection to the first gel is 20:1, so as to obtain a solution containing the first polysaccharide polymer (sol containing the first polysaccharide polymer).
(2.2) Preparation of a solution comprising the second polysaccharide Polymer
(2.2.1) Weighing a certain amount of carboxymethyl chitosan (second crosslinking monomer) with viscosity of 37.2 mPas, preparing a solution with mass concentration of 2% by using water for injection (second solvent), stirring by using an emulsifying machine, placing the solution in 2-8 ℃ to swell for more than 15 hours, stirring again after the swelling is finished, and stirring for about 30 minutes to obtain a second solution;
(2.2.2) adding a second crosslinking agent 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) to the second solution, wherein the mass ratio of the second crosslinking agent to the second solution is 0.1:1000, and stirring for 5 minutes by using an emulsifying machine to obtain a solution containing a second polysaccharide polymer.
(2.2.3) Preparation of film Forming liquid
Blending the solution containing the first polysaccharide polymer and the solution containing the second polysaccharide polymer according to the mass ratio of 2:1, homogenizing and stirring by a vacuum emulsifying machine, wherein the homogenizing rotating speed is set to 1500rpm, the stirring rotating speed is set to 45rpm, the homogenizing time is set to 5min, and the stirring time is set to 8min, so as to obtain the film forming liquid.
(3) Double-layer composite
The low-density polyethylene film is paved and closely attached on the bottom surface of the die for being paved, the smooth surface of the material of the repairing layer obtained in the step (1.2) is paved on the low-density polyethylene film upwards, and the bottom surface of the die is completely covered;
(3.2) adding the film forming liquid obtained in the step (2.2.3) onto the material of the repairing layer in the step (3.1), and flatly laying the film forming liquid on the material of the repairing layer in a gravity casting mode, standing for more than 5 hours to form an intermediate with a double-layer structure, wherein the ratio of the mass of the film forming liquid to the area of the material of the repairing layer contacted with the film forming liquid is 2.0 g/10 cm2;
(3.3) carrying out vacuum freeze drying treatment on the double-layer structure formed in the step (3.2);
And (3.4) pressing and cutting the dried sample in the step (3.3) to form the required thickness and size, packaging and sterilizing at a terminal to obtain the final product artificial biomembrane patch, wherein the final product artificial biomembrane patch comprises a repairing layer and an anti-blocking layer arranged on one side of the repairing layer, the anti-blocking layer is formed by the film forming liquid, the anti-blocking layer has a porous structure, the porosity of the anti-blocking layer is 77.03%, the average crosslinking degree of a first polysaccharide polymer (carboxymethyl chitosan) in the anti-blocking layer is 58.14%, the average crosslinking degree of a second polysaccharide polymer (carboxymethyl chitosan) in the anti-blocking layer is 11.25%, the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the anti-blocking layer is 2:1, and the mass of the anti-blocking layer on the repairing layer per unit area is 2.6mg/cm2.
Example 2
The preparation method of the artificial biological membrane patch is basically the same as that of example 1, except that in the preparation of the solution containing the first polysaccharide polymer, the mass ratio of the first crosslinking agent to the first solution in the step (2.1.2) is 2:1000, the porosity of the anti-blocking layer of the artificial biological membrane patch obtained in the step (3.4) is 71.24%, and the average crosslinking degree of the first polysaccharide polymer in the anti-blocking layer is 64.25%.
Example 3
The artificial biomembrane patch was prepared substantially as in example 1 except that in the preparation of the solution containing the first polysaccharide polymer, the mass ratio of the first crosslinking agent to the first solution in the step (2.1.2) was 3:1000, the porosity of the anti-blocking layer of the artificial biomembrane patch obtained in the step (3.4) was 64.07%, and the average crosslinking degree of the first polysaccharide polymer in the anti-blocking layer was 81.95%.
Example 4
The artificial biomembrane patch was prepared substantially as in example 1 except that in the preparation of the solution containing the second polysaccharide polymer, the mass ratio of the second crosslinking agent to the second solution in the step (2.2.2) was 0.3:1000, the porosity of the anti-blocking layer of the artificial biomembrane patch obtained in the step (3.4) was 73.25%, and the average crosslinking degree of the second polysaccharide polymer in the anti-blocking layer was 17.65%.
Example 5
The artificial biomembrane patch was prepared substantially as in example 1 except that in the preparation of the solution containing the second polysaccharide polymer, the mass ratio of the second crosslinking agent to the second solution in the step (2.2.2) was 0.4:1000, the porosity of the anti-blocking layer of the artificial biomembrane patch obtained in the step (3.4) was 70.35%, and the average crosslinking degree of the second polysaccharide polymer in the anti-blocking layer was 23.11%.
Example 6
The preparation method of the artificial biological membrane patch is basically the same as that of example 1, except that in the preparation of the film forming solution in step (2.2.3), the solution containing the first polysaccharide polymer and the solution containing the second polysaccharide polymer are blended according to a mass ratio of 1:1, the porosity of the anti-adhesion layer of the artificial biological membrane patch obtained in step (3.4) is 82.35%, and the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the anti-adhesion layer is 1:1.
Example 7
The preparation method of the artificial biological membrane patch is basically the same as that of example 1, except that in the preparation of the film forming solution in step (2.2.3), the solution containing the first polysaccharide polymer and the solution containing the second polysaccharide polymer are blended according to a mass ratio of 4:1, the porosity of the anti-adhesion layer of the artificial biological membrane patch obtained in step (3.4) is 68.35%, and the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the anti-adhesion layer is 4:1.
Example 8
The preparation method of the artificial biological membrane patch is basically the same as that of example 1, except that in the step (3.2), the ratio of the mass of the film forming liquid to the area of the material of the repair layer contacting the film forming liquid is 2.2g:10cm2, and in the artificial biological membrane patch obtained in the step (3.4), the porosity of the anti-adhesion layer is 78.35%, and the mass of the anti-adhesion layer on the repair layer per unit area is 2.8mg/cm2.
Example 9
The preparation method of the artificial biological membrane patch is basically the same as that of example 1, except that in the step (3.2), the ratio of the mass of the film forming liquid to the area of the material of the repair layer contacting the film forming liquid is 2.6g:10cm2, and in the artificial biological membrane patch obtained in the step (3.4), the porosity of the anti-adhesion layer is 79.51%, and the mass of the anti-adhesion layer on the repair layer per unit area is 3.1mg/cm2.
Example 10
The preparation method of the artificial biological membrane patch is basically the same as that of example 1, except that in the step (3.2), the ratio of the mass of the film forming liquid to the area of the material of the repair layer contacting the film forming liquid is 3g:10cm2, and in the artificial biological membrane patch obtained in the step (3.4), the porosity of the anti-adhesion layer is 80.32%, and the mass of the anti-adhesion layer on the repair layer per unit area is 3.3mg/cm2.
Example 11
The preparation method of the artificial biological membrane patch is basically the same as that of example 1, except that in the step (3.2), the ratio of the mass of the film forming liquid to the area of the material of the repair layer contacting the film forming liquid is 3.2g:10cm2, and in the artificial biological membrane patch obtained in the step (3.4), the porosity of the anti-adhesion layer is 83.22%, and the mass of the anti-adhesion layer on the repair layer per unit area is 3.6mg/cm2.
Comparative example 1
The preparation method of the artificial biological membrane patch is basically the same as in example 1, except that in the preparation of the solution containing the first polysaccharide polymer, the mass ratio of the first crosslinking agent to the first solution in the step (2.1.2) is 0.5:1000, the porosity of the anti-blocking layer of the artificial biological membrane patch obtained in the step (3.4) is 89.35%, and the average crosslinking degree of the first polysaccharide polymer in the anti-blocking layer is 35.15%.
Comparative example 2
The preparation method of the artificial biological membrane patch is basically the same as that of example 1, except that in the preparation of the solution containing the first polysaccharide polymer, the mass ratio of the first crosslinking agent to the first solution in the step (2.1.2) is 4:1000, the porosity of the anti-blocking layer of the artificial biological membrane patch obtained in the step (3.4) is 61.28%, and the average crosslinking degree of the first polysaccharide polymer in the anti-blocking layer is 92.25%.
Comparative example 3
The preparation method of the artificial biological membrane patch is basically the same as in example 1, except that in the preparation of the solution containing the second polysaccharide polymer, the mass ratio of the second crosslinking agent to the second solution in the step (2.2.2) is 0.05:1000, the porosity of the anti-blocking layer of the artificial biological membrane patch obtained in the step (3.4) is 83.26%, and the average crosslinking degree of the second polysaccharide polymer in the anti-blocking layer is 4.23%.
Comparative example 4
The artificial biomembrane patch was prepared substantially as in example 1 except that in the preparation of the solution containing the second polysaccharide polymer, the mass ratio of the second crosslinking agent to the second solution in the step (2.2.2) was 0.5:1000, the porosity of the anti-blocking layer of the artificial biomembrane patch obtained in the step (3.4) was 73.41%, and the average crosslinking degree of the second polysaccharide polymer in the anti-blocking layer was 32.15%.
Comparative example 5
The preparation method of the artificial biological membrane patch is basically the same as that of example 1, except that in the preparation of the film forming solution in step (2.2.3), the solution containing the first polysaccharide polymer and the solution containing the second polysaccharide polymer are blended according to a mass ratio of 0.5:1, the porosity of the anti-blocking layer of the artificial biological membrane patch obtained in step (3.4) is 93.21%, and the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the anti-blocking layer is 0.5:1.
Comparative example 6
The preparation method of the artificial biological membrane patch is basically the same as that of example 1, except that in the preparation of the film forming solution in step (2.2.3), the solution containing the first polysaccharide polymer and the solution containing the second polysaccharide polymer are blended according to a mass ratio of 5:1, the porosity of the anti-adhesion layer of the artificial biological membrane patch obtained in step (3.4) is 58.14%, and the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the anti-adhesion layer is 5:1.
Test method
(1) Average degree of crosslinking test of first polysaccharide Polymer and second polysaccharide Polymer
(1.1) Preparation of reagents:
An acetic acid buffer solution with pH of 5.4 and concentration of 2mol/L is prepared by weighing 86mL of 2mol/L sodium acetate solution and adding 14mL of 2mol/L acetic acid for mixing. The corrected pH was checked with a pH meter.
An ninhydrin color solution was prepared, and 85mg of ninhydrin and 15mg of ninhydrin were weighed and dissolved in 10mL of ethylene glycol monomethyl ether.
Preparation of Indantrione 5g of ninhydrin was weighed and dissolved in 125mL of boiling purified water to give a yellow solution. 5g of vitamin C was dissolved in 250mL of warm purified water, and the vitamin C solution was added dropwise to the ninhydrin solution with stirring, and precipitation was continued. Stirring for 15min, cooling to 4deg.C in refrigerator, filtering to obtain precipitate, washing with cold water for 3 times, and drying in a dryer.
Preparing 60% ethanol, namely transferring 60mL of absolute ethanol, and adding purified water to a volume of 100mL.
(1.2) Preparation of a Standard Curve, an amino acid standard solution with a concentration of 2.5mmol/L was taken as a stock solution. The standard curve was prepared as in table 1 below.
TABLE 1
(1.3) Sample preparation, namely precisely weighing 300mg of sample before crosslinking and 300mg of sample after crosslinking into a 50mL test tube respectively, adding water for dissolution and fixing the volume.
(1.4) Developing, namely transferring 1mL of each standard substance solution and each sample solution into a test tube, respectively adding 1mL of acetic acid buffer solution with the pH of 5.4 and the concentration of 2mol/L and 1mL of ninhydrin developing solution, uniformly mixing, heating in a boiling water bath at 100 ℃ for 15min, and cooling by tap water. After 5min, 3mL of 60% ethanol was added for dilution.
(1.5) Measurement was performed at a wavelength of 570nm using an ultraviolet spectrophotometer. And drawing a standard curve by taking an OD570nm value as an ordinate and the concentration of an amino acid standard substance as an abscissa, calculating to obtain the concentration of the amino acid in the sample, and calculating to obtain the average crosslinking degree through the amino acid content change before and after crosslinking of the carboxymethyl chitosan solution.
Wherein C1 -the concentration of sample amino acid before crosslinking (mmol/L);
c2 -sample amino acid concentration (mmol/L) after crosslinking;
m1 -sample weight before crosslinking (mg);
m2 -sample weight after crosslinking (mg).
When testing the average degree of crosslinking of the first polysaccharide polymer, the pre-crosslinking sample refers to a solution, e.g., a first solution, comprising the first crosslinking monomer prior to crosslinking, and the post-crosslinking sample refers to a solution comprising the first polysaccharide polymer after crosslinking.
When testing the average degree of crosslinking of the second polysaccharide polymer, the pre-crosslinking sample refers to a solution, e.g., a second solution, comprising the second crosslinking monomer prior to crosslinking, and the post-crosslinking sample refers to a solution comprising the second polysaccharide polymer after crosslinking.
(2) Porosity test of anti-blocking layer
The porosity of the anti-blocking layer is detected by referring to GB/T33052-2016 method for measuring porosity of microporous functional film hexadecane absorption method.
Taking an artificial biological membrane patch sample, measuring the length a, the width b and the thickness c, precisely weighing the mass of the sample, recording as m1, immersing the sample in a container filled with absolute ethyl alcohol for 30min, taking out the sample, wiping off the absolute ethyl alcohol on the surface, rapidly weighing the mass of the sample filled with absolute ethyl alcohol, recording as m2, and calculating the porosity P of the anti-blocking layer as follows:
wherein m1 is the dry weight of the sample, and the unit is g;
m2 is the mass of the sample after being filled with absolute ethyl alcohol, and the unit is g;
a is the length of the sample in mm;
b is the width of the sample in mm;
c is the thickness of the sample in mm;
ρ is the density of absolute ethanol, 0.79g/cm3.
Test example 1 measurement of interlayer bonding Strength
Referring to fig. 1, the artificial biomembrane patch samples prepared in examples 1 to 11 and comparative examples 1 to 6 were cut into strip-shaped test pieces having a width of 15.0±0.1mm and a length of 30mm, 3M double-sided tape was tightly adhered to the surface of the repair layer of the test piece, one end of the test piece was peeled off by 10mm in advance, and both sides of the peeled off were respectively clamped on the upper and lower clamps 1 and 2 of the tester, so that the longitudinal axes of the peeled off portions 3 of the test piece were coincident with the central lines of the upper and lower clamps 1 and 2, and were suitably elastic. In the test, the non-peeled portion 4 of the sample was T-shaped with respect to the stretching direction, a test speed of 300.+ -.50 mm/min was selected, and the peeling force during peeling of the sample was recorded, and the interlayer bonding strength was calculated according to the following formula, interlayer bonding strength (kN/m) =maximum peeling force (N)/sample width (mm), and the test results are shown in Table 2.
TABLE 2
As can be seen from table 2, compared with comparative examples 1 to 5, the interlayer bonding strength between the repair layer and the anti-blocking layer of the artificial biomembrane patch prepared in examples 1 to 11 is higher, which means that examples 1 to 11 control the average degree of crosslinking of the first polysaccharide polymer and the average degree of crosslinking of the second polysaccharide polymer in the anti-blocking layer, and the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer is within a reasonable range, so that the bonding force between the first polysaccharide polymer and the second polysaccharide polymer in the anti-blocking layer and the repair layer is improved, and the improvement of the bonding force can reduce scar hyperplasia, thereby improving the anti-blocking effect of the artificial biomembrane patch.
Test example 2 cytotoxicity test
Samples of the artificial biofilm patches prepared in example 1, example 3, example 5, example 7, comparative example 2, comparative example 4 and comparative example 6 were taken for cytotoxicity test detection.
Cell preparation rat fibroblasts were resuscitated using MEM medium containing 10% FBS (fetal bovine serum) and cultured at 37℃under 5% CO2 at saturated humidity for 2-3 passages to the cell-geminate growth phase, and the cells were digested with trypsin, collected and adjusted to a cell concentration of 1X 105 cells/mL for the following experiment.
Sample preparation samples were processed at a leaching rate of 6cm2/mL, leaching medium MEM medium 0.5mL, leaching conditions of 37℃and shaking at 125rpm for 72h.
Negative samples were prepared by shaking and leaching in MEM medium containing 10% FBS at 37℃for 72h in a 50mL sterile centrifuge tube.
Positive sample preparation MEM complete medium containing 10% dmso (dimethyl sulfoxide) was prepared.
Blank sample preparation MEM medium with 10% FBS.
Experimental methods cell suspensions were added to 96-well plates at 100. Mu.L per well for a total of 1X 104 cells/well, 37℃and 5% CO2 cultured for 24h. After the culture, the medium in the plate was discarded, and the sample extracts (100%, 50%,25% and 12.5% of four dose groups) were added, respectively, and the negative control, the blank control and the positive control, each group of 6 wells, were placed in a 37 ℃ and 5% CO2 saturated humidity incubator. After the completion of the 24-hour incubation, the plate medium was discarded, 50. Mu.L (1 mg/mL) of MTT (thiazolyl blue) stain was added to each well, and the mixture was placed in a 37℃and 5% CO2 saturated humidity incubator for incubation for 2 hours. After the culture is finished, the liquid in the culture plate is discarded, 100 mu L of isopropanol is added into each hole, the mixture is vibrated and mixed uniformly for 30min in a dark place, and the mixture is put into an enzyme-labeled instrument with 570nm as a detection wavelength and 650nm as a reference wavelength to detect absorbance.
Analysis of experimental results the relative proliferation rate of cells was calculated according to the following formula from the absorbance mean of each group.
Relative increment rate (%) =100×ode/ODb
ODe-the average value of the 100% leaching liquor optical density of the test sample;
Odb—blank optical density average;
The experimental raw data were processed to assess the toxicity of the materials according to tables 3 and 4.
TABLE 3 cytotoxicity reaction fractionation criteria
TABLE 4 cytotoxicity reaction fractionation criteria
TABLE 5 relative increment rate and toxicity rating
As can be seen from tables 3 to 5 and fig. 2, comparative examples 2, 4 and 6 exhibit significant cytotoxicity reaction due to the average crosslinking degree of the first polysaccharide polymer, the average crosslinking degree of the second polysaccharide polymer or the mass ratio of the first polysaccharide polymer in the anti-blocking layer in the artificial biofilm patches prepared in comparative examples 2, 4 and 6 being too high. Examples 1, 3, 5 and 7 did not exhibit a cytotoxic reaction, and examples 2, 4, 6, 8-11 did not exhibit a cytotoxic reaction, demonstrating that the artificial biofilm patches prepared in examples 1-11 were biosafety.
Test example 3 anti-leakage test
Anti-leakage test detection was performed by taking the artificial biofilm patch samples prepared in example 1, example 3, example 5, example 7, example 8, example 9, example 10, comparative example 2, comparative example 4, comparative example 6 and comparative example 8.
The sample is fixed in an antiseep detection device shown in figure 1 of the specification drawing of Chinese patent CN219552202U, physiological saline in a cylindrical funnel is dyed with a methylene blue solution for convenient observation, and the sample is pressed by applying 40mmHg pressure to a liquid storage bag according to an inflatable part and corresponding pressure value is recorded by a pressure gauge. The test results are shown in FIG. 3.
FIG. 3 shows that the artificial biomembrane patches prepared in examples 1,3, 5, 7, 8, 9, 10 and 11 are capable of preventing leakage by showing various degrees of leakage of the Melan solution under a pressure of 40mmHg in each of comparative examples 2, 4 and 6.
Test example 4 fibroblast proliferation assay
The artificial biofilm patch sample prepared in example 1 and a sample of a commercially available like product (simple extracellular matrix material) having only a repair layer and no anti-adhesion layer were taken for fibroblast proliferation test.
Cell culture, in which cells are inoculated in a culture solution containing 10% fetal bovine serum for culture, and placed in a cell incubator with 37 ℃ and 5% CO2. Cells in logarithmic growth phase were taken and passaged with 0.25% pancreatin. And taking 3-5-generation cells for further testing.
Cell proliferation, namely trimming a sample, placing the trimmed sample into a 24-well plate, taking P3 generation cells with good growth, inoculating 105 cells onto the surface of the sample (3 cm x 4 cm), setting three parallel samples, respectively incubating for 1d, 2d, 3d, 5d and 7d, adding 20 mu L of MTT solution (5 mg/mL) into each hole, incubating for 3-4 h in an incubator, taking out, slightly sucking liquid in the well plate by using a pipetting gun, adding 150 mu L of DMSO into each hole, and measuring absorbance at 570nm by using an enzyme-labeled instrument within 15 minutes. The test results are shown in fig. 4.
As can be seen from fig. 4, the artificial biomembrane patch prepared in example 1 inhibits fibroblast proliferation after implantation, can play a role of mechanical barrier and reduce the effect of surgical adhesion, and further demonstrates that the anti-adhesion effect of the artificial biomembrane patch prepared in the application is excellent.
Test example 5 microstructure analysis
The three-dimensional pore structure of the artificial biofilm patch sample prepared in example 1 was analyzed by Micro-CT. The sample is placed in a detection instrument, detection parameters of 60KV voltage, 80 MuA current, 700s integration time, 1440 projection number and 2 Mum resolution are set, and the whole area of the sample is selected and analyzed for porosity.
As can be seen from fig. 5, the three-dimensional structure of the artificial biomembrane patch prepared in example 1 shows a clear layered structure, the upper layer is an anti-adhesion layer and is in a loose porous shape, the lower layer is a repair layer containing extracellular matrix, the arrangement is relatively dense, and the transition layer between the upper layer and the lower layer is formed between the first polysaccharide polymer and/or the second polysaccharide polymer in the anti-adhesion layer and the extracellular matrix in the repair layer through electrostatic action and secondary bonds.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the application, which are described in detail and are not to be construed as limiting the scope of the claims. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. The scope of the application is, therefore, indicated by the appended claims, and the description may be intended to interpret the contents of the claims.