CN118079088A - Double-layer collagen membrane and preparation method and application thereof - Google Patents
Double-layer collagen membrane and preparation method and application thereof Download PDFInfo
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- Materials For Medical Uses (AREA)
Abstract
The invention provides a double-layer collagen film, a preparation method and application thereof, wherein the double-layer collagen film comprises an upper collagen layer and a lower collagen layer which are sequentially laminated; the upper collagen layer is a crosslinked collagen layer; the lower collagen layer is an uncrosslinked collagen layer; the double-layer collagen membrane has a mesh structure which is penetrated up and down. In the invention, the double-layer collagen membrane has higher mechanical strength and can provide better mechanical support; can provide nutrition for the wound surface, promote nutrition supply, and facilitate cell ingrowth and tissue reconstruction; the material can also load the medicine, release the medicine continuously, prolong the action period of the medicine, and in addition, the material has good biocompatibility and good water absorption, and can meet the requirements of suturing and filling.
Description
Technical Field
The invention belongs to the technical field of repair materials, and particularly relates to a double-layer collagen membrane and a preparation method and application thereof.
Background
At present, there are a great number of technical solutions proposed for reconstructing and repairing the tissue around the dermis defect, and the commercial technical solutions mainly include a natural dermis repairing material prepared by a decellularization technology and an artificial dermis repairing material synthesized by purified collagen or other degradable materials.
However, natural dermis restoration materials prepared by decellularization techniques, although having higher mechanical properties and longer degradation times, may present a risk of carrying viruses, immunogenicity. The artificial dermis repairing material has the advantages of controllable performance, proper pore diameter and good pore penetrability, but has low mechanical property, cannot meet the clinical suturing, moving and other operations, and often needs a high-strength polymer layer as an auxiliary layer to provide mechanical support. If CN103191085A discloses a double-layer composite regeneration membrane, a compact layer is compounded on the loose layer, the compact layer is formed by laminating and pressing degradable polymer fiber mesh and collagen membrane, but the compact layer is of a compact structure, does not have a hole structure, only provides mechanical support, and cannot play a role in promoting tissue reconstruction.
In another example, CN107412870a discloses a collagen-based skin repair material with a double-layer porous structure, wherein the double-layer collagen-based material is crosslinked by EDC/NHS, the upper layer is a collagen film layer with a nano-pore structure obtained after collagen solution is air-dried, the nano-pore structure is still not suitable for the growth of cells, and the double-layer crosslinking can cause the degradation speed of the collagen film to be slower, so that nutrition can not be provided for wound surfaces in time.
Therefore, the method has good mechanical strength, and can meet the clinical suturing, moving and other operations; has good pore structure, and can promote cell ingrowth and tissue reconstruction; can provide early nutrition supply for the wound surface and promote the regeneration of the wound surface; the collagen repairing material which can also load and continuously release medicines and has the functions of bacteriostasis, inflammation reduction and the like is a problem to be solved in the field.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide a double-layer collagen membrane, and a preparation method and application thereof. The double-layer collagen membrane has higher mechanical strength and can provide better mechanical support; can provide nutrition for the wound surface, promote nutrition supply, and facilitate cell ingrowth and tissue reconstruction; the material can also load the medicine, release the medicine continuously, prolong the action period of the medicine, and in addition, the material has good biocompatibility and good water absorption, and can meet the requirements of suturing and filling.
To achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a bilayer collagen membrane comprising an upper collagen layer and a lower collagen layer laminated in sequence; the upper collagen layer is a crosslinked collagen layer; the lower collagen layer is an uncrosslinked collagen layer; the double-layer collagen membrane has a mesh structure which is penetrated up and down.
In the invention, the mesh structure which is vertically communicated is beneficial to promoting nutrition supply, and the longitudinal aperture ratio of the collagen membrane is increased by the through pore canal, so that the growth of cells and blood vessels is further guided, and the tissue reconstruction is promoted; the lower collagen layer is an uncrosslinked collagen layer, can be rapidly degraded, and the degradation products are amino acid components, so that nutrition supply can be provided for the wound surface, and the regeneration of the wound surface can be promoted; the upper layer collagen film is a crosslinked collagen film, so that the medicine can be loaded, and the treatment effect is improved; the collagen membrane has higher mechanical strength, can provide better mechanical support, has better biocompatibility and high water absorbability, and can meet the requirements of suturing and filling.
Preferably, the apparent density of the upper collagen layer is 35 to 120mg/cm 3, for example, 35mg/cm3、40mg/cm3、45mg/cm3、50mg/cm3、55mg/cm3、60mg/cm3、65mg/cm3、70mg/cm3、75mg/cm3、80mg/cm3、85mg/cm3、90mg/cm3、95mg/cm3、100mg/cm3、105mg/cm3、110mg/cm3、115mg/cm3、120mg/cm3 or the like.
In the invention, the upper collagen layer with larger density is used for loading the medicine, so that the slow release of the medicine can be realized.
Preferably, the thickness of the upper collagen layer is 0.5 to 1.5mm, for example, 0.5mm, 0.6mm, 0.8mm, 1mm, 1.2mm, 1.4mm, 1.5mm, etc. may be used.
Preferably, the upper collagen layer is loaded with an antibacterial agent.
Preferably, the antibacterial agent comprises any one or a combination of at least two of tannic acid, polyphosphate, polyhexamethylene biguanide hydrochloride, polypeptide or bletilla striata polysaccharide.
According to the invention, by loading the antibacterial drugs, the antibacterial and anti-inflammatory effects can be achieved, and wound repair is facilitated.
Preferably, the apparent density of the lower collagen layer is 15 to 80mg/cm 3, for example, 15mg/cm3、20mg/cm3、25mg/cm3、30mg/cm3、35mg/cm3、40mg/cm3、45mg/cm3、50mg/cm3、55mg/cm3、60mg/cm3、65mg/cm3、70mg/cm3、75mg/cm3、80mg/cm3 or the like.
Preferably, the thickness of the lower collagen layer is 0.3 to 1.5mm, and may be, for example, 0.3mm, 0.4mm, 0.5mm, 0.6mm, 0.8mm, 1mm, 1.2mm, 1.4mm, 1.5mm, etc.
Preferably, the mesh structure comprises at least one of a linear mesh, a diamond mesh or a circular mesh.
Preferably, the length of the linear mesh is 1-5 mm, for example 1mm、1.2mm、1.4mm、1.6mm、1.8mm、2mm、2.2mm、2.4mm、2.6mm、2.8mm、3mm、3.2mm、3.4mm、3.6mm、3.8mm、4mm、4.2mm、4.4mm、4.6mm、4.8mm、5mm; the areas of the diamond-shaped mesh and the round mesh are respectively and independently 0.03-3.20 mm 2, and can be 0.03mm2、0.04mm2、0.06mm2、0.08mm2、0.1mm2、0.2mm2、0.4mm2、0.6mm2、0.8mm2、1mm2、1.2mm2、1.4mm2、1.6mm2、1.8mm2、2mm2、2.2mm2、2.4mm2、2.6mm2、2.8mm2、3mm2、3.2mm2, for example.
Preferably, the distance between the two adjacent rows of meshes is 0.2-2 mm, for example, 0.2mm, 0.4mm, 0.6mm, 0.8mm, 1mm, 1.2mm, 1.4mm, 1.6mm, 1.8mm, 2mm and the like can be used.
Preferably, the porosity of the bilayer collagen membrane is not less than 75%, for example, 75%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, etc.
In the invention, the collagen membrane is of a porous bracket structure.
Preferably, the materials of the upper and lower collagen layers each independently comprise any one or a combination of at least two of type I collagen, type II collagen, or type III collagen.
In the invention, the degradation time of the lower collagen layer is less than or equal to 24h, and can be 1h, 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h and the like.
In a second aspect, the present invention provides a method for preparing a bilayer collagen membrane according to the first aspect, the method comprising the steps of:
And laminating the upper collagen layer and the lower collagen layer, and preparing meshes to obtain the double-layer collagen film.
Preferably, the preparation method of the upper collagen layer comprises the following steps:
mixing the upper collagen solution, phosphate buffer solution and cross-linking agent, regulating the pH value of the system to 7.5-8 after cross-linking, and then incubating to obtain gel; and washing and extruding the gel to obtain the upper collagen layer.
Preferably, the weight fraction of collagen in the upper collagen solution is 0.3 to 0.8%, for example, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, etc.
Preferably, the upper collagen solution further comprises an antibacterial agent.
Preferably, the mass ratio of the upper collagen solution to the phosphate buffer is (8-10): 1, for example, 8:1, 8.5:1, 9:1, 9.5:1, 10:1, etc.
In the present invention, the phosphate buffer solution comprises a 10X PBS solution.
Preferably, the cross-linking agent is glutaraldehyde.
Preferably, the mass concentration of the crosslinking agent in the system is 0.01 to 0.1%, for example, may be 0.01%, 0.02%, 0.04%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, or the like.
Preferably, the temperature of the crosslinking is 0 to 10 ℃, for example, 2 ℃,4 ℃,6 ℃, 8 ℃, 10 ℃ and the like; the time is 0.5 to 3 hours, and may be, for example, 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, etc.
In the invention, sodium hydroxide solution can be used for regulating the pH value of the system.
Preferably, the incubation temperature is 35 to 38 ℃, for example, 35 ℃, 36 ℃, 37 ℃, 38 ℃ and the like; the time is 40-100 min, for example, 40min, 50min, 60min, 70min, 80min, 90min, 100min, etc.
In the present invention, the washing comprises: the gel obtained was washed sequentially with PBS buffer, 75% volume fraction ethanol and purified water.
In the present invention, the squeezing includes placing the washed gel on a water absorbing material, placing a 200 mesh nylon net on the gel, and placing a weight of 50 to 120g (for example, 50g, 60g, 70g, 80g, 90g, 100g, 110g, 120g, etc.) on the nylon net, and squeezing the hydrogel.
Preferably, the preparation method of the lower collagen layer comprises the following steps:
coating a lower collagen solution on one surface of the upper collagen layer, and freeze-drying to obtain a laminated upper collagen layer and lower collagen layer.
Preferably, the weight fraction of collagen in the lower collagen solution is 0.3 to 0.8%, for example, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, etc.
Preferably, the apparatus for preparing a mesh comprises a netmaker and/or a laser.
In a third aspect, the present invention provides a dermis restoration material comprising a bilayer collagen membrane according to the first aspect.
The numerical ranges recited herein include not only the recited point values, but also any point values between the recited numerical ranges that are not recited, and are limited to, and for the sake of brevity, the invention is not intended to be exhaustive of the specific point values that the recited range includes.
Compared with the prior art, the invention has the beneficial effects that:
(1) The double-layer collagen membrane provided by the invention has higher mechanical strength, can provide better mechanical support when used for tissue filling, can meet the requirements of suturing and filling, and has better biocompatibility and high water absorbability.
(2) The double-layer collagen membrane provided by the invention has a through mesh structure, the through pore canal can promote nutrition supply, and meanwhile, the longitudinal opening rate of the collagen membrane is increased by the through pore canal, so that cells and blood vessels can be further guided to grow in, and tissue reconstruction is promoted.
(3) The collagen layer under the double-layer collagen film provided by the invention is an uncrosslinked collagen layer, can be rapidly degraded, and the degradation products are amino acid components, so that nutrition supply is provided for the wound surface, and the regeneration of the wound surface is further promoted.
(4) The collagen layer on the double-layer collagen film provided by the invention is a crosslinked collagen layer, so that the medicine components can be loaded, and the medicine slow release is realized.
Drawings
FIG. 1 is a schematic diagram of a double-layer collagen membrane structure according to the present invention;
Wherein, 1-an upper collagen layer; 2-a lower collagen layer; 3-a mesh structure penetrating up and down;
FIG. 2 is a graph showing the degradation results of the bilayer collagen membrane provided in example 1;
wherein, FIG. 2-1 is an effect graph of degradation for 0 h; FIG. 2-2 is a graph showing the effect of degradation for 7 h; FIGS. 2-3 are graphs showing the effect of degradation for 11 h; FIGS. 2-4 are graphs showing the effect of degradation 22 h;
FIG. 3 is a graph showing the degradation results of the single-layer collagen film provided in comparative example 1;
wherein, FIG. 3-1 is an effect graph of degradation for 0 h; FIG. 3-2 is a graph showing the effect of degradation for 7 h; FIG. 3-3 is a graph showing the effect of degradation for 11 h; FIGS. 3-4 are graphs showing the effect of degradation 22 h;
FIG. 4 is a graph showing the degradation results of the bilayer collagen membrane provided in comparative example 3;
wherein, FIG. 4-1 is an effect graph of degradation for 0 h; FIG. 4-2 is a graph showing the effect of degradation for 7 h; FIG. 4-3 is a graph showing the effect of degradation for 11 h; FIGS. 4-4 are graphs showing the effect of degradation 22 h;
FIG. 5 is an SEM image of a bilayer collagen membrane provided in example 1;
FIG. 5-1 is an SEM image of the upper collagen layer of the double-layered collagen film provided in example 1 when the double-layered collagen film is not net-laid; FIG. 5-2 is an SEM image of the collagen layer of the double-layered collagen film provided in example 1 without screening; FIGS. 5-3 are SEM images of bilayer collagen films provided in example 1; FIGS. 5-4 are side SEM images of a bilayer collagen membrane provided by example 1;
FIG. 6 is an SEM image of a bilayer collagen membrane provided in example 2;
FIG. 6-1 is an SEM image of the upper collagen layer of the double-layered collagen film provided in example 2 when the double-layered collagen film is not net-laid; FIG. 6-2 is an SEM image of the collagen layer of the double-layered collagen film provided in example 2 without screening;
Fig. 7 is a side SEM image of the bilayer collagen membrane provided in example 3.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
The materials used in the invention are as follows:
collagen used for the upper collagen layer: type I collagen;
Collagen used for the lower collagen layer: type I collagen;
The upper collagen solution and the lower collagen solution are prepared by dissolving collagen in pure water.
Example 1
The present embodiment provides a double-layer collagen film, the structural schematic diagram of which is shown in fig. 1, comprising an upper collagen layer 1 and a lower collagen layer 2 which are sequentially laminated; the upper collagen layer is a crosslinked collagen layer; the lower collagen layer is an uncrosslinked collagen layer; the double-layer collagen membrane is provided with a mesh structure 3 which penetrates up and down.
The embodiment provides a preparation method of a double-layer collagen membrane, which comprises the following steps:
(1) Preparing an upper collagen solution with the weight percent of 0.5;
(2) Adding 10X PBS (the mass ratio of the collagen solution to the 10X PBS is 9:1) into the upper collagen solution, then adding glutaraldehyde to ensure that the concentration of glutaraldehyde is 0.01wt%, stirring for 30min at 4 ℃, adjusting the pH to 7.5-8.0 by using sodium hydroxide solution, pouring the defoamed solution into a tray according to 0.3-0.5 g/cm 2 after defoamed treatment, and incubating for 60min at 37 ℃ to form gel;
(3) And (3) washing the gel obtained in the step (2) with PBS buffer solution for multiple times, then washing with 75% ethanol by volume fraction, finally washing with purified water, taking out the gel after washing, placing the gel on a filter paper stack, placing a 200-mesh nylon net on the gel, placing 80g of heavy objects on the nylon net, and extruding the hydrogel to obtain the upper collagen layer.
(4) Coating a layer of 0.5wt% lower collagen solution on one surface of the upper collagen layer obtained in the step (3), then pre-freezing in an ultralow temperature refrigerator at-60 ℃, transferring to a freeze dryer for freeze drying, and screening (linear mesh) the collagen film by a screening machine to obtain the double-layer collagen film.
Example 2
The present embodiment provides a double-layer collagen film, the structural schematic diagram of which is shown in fig. 1, comprising an upper collagen layer 1 and a lower collagen layer 2 which are sequentially laminated; the upper collagen layer is a crosslinked collagen layer; the lower collagen layer is an uncrosslinked collagen layer; the double-layer collagen membrane is provided with a mesh structure 3 which penetrates up and down.
The embodiment provides a preparation method of a double-layer collagen membrane, which comprises the following steps:
(1) Preparing 0.5wt% of collagen-bletilla striata polysaccharide solution (the mass ratio of collagen to bletilla striata polysaccharide is 10:1);
(2) Adding 10X PBS (the mass ratio of the collagen solution to the 10X PBS is 9:1) into the upper collagen-bletilla striata polysaccharide solution, then adding glutaraldehyde to ensure that the concentration of glutaraldehyde is 0.01wt%, stirring for 30min at 4 ℃, adjusting the pH to 7.5-8.0 by using a sodium hydroxide solution, pouring the defoamed solution into a tray according to 0.3-0.5 g/cm 2 after defoamed treatment, and incubating for 60min at 37 ℃ to form gel;
(3) And (3) washing the gel obtained in the step (2) with PBS buffer solution for multiple times, then washing with 75% ethanol by volume fraction, finally washing with purified water, taking out the gel after washing, placing the gel on a filter paper stack, placing a 200-mesh nylon net on the gel, placing 80g of heavy objects on the nylon net, and extruding the hydrogel to obtain the upper collagen layer loaded with the antibacterial drugs.
(4) Coating one surface of the upper collagen layer obtained in the step (3) with a layer of 0.5wt% lower collagen solution, then freezing in an ultralow temperature refrigerator at-60 ℃, transferring to a freeze dryer for freeze drying, and screening (linear mesh) the collagen film by a screening machine to obtain the double-layer collagen film.
Example 3
The present embodiment provides a double-layer collagen film, the structural schematic diagram of which is shown in fig. 1, comprising an upper collagen layer 1 and a lower collagen layer 2 which are sequentially laminated; the upper collagen layer is a crosslinked collagen layer; the lower collagen layer is an uncrosslinked collagen layer; the double-layer collagen membrane is provided with a mesh structure 3 which penetrates up and down.
The embodiment provides a preparation method of a double-layer collagen membrane, which comprises the following steps:
(1) Preparing an upper collagen solution with the weight percent of 0.8;
(2) Adding 10X PBS (the mass ratio of the collagen solution to the 10X PBS is 9:1) into the upper collagen solution, then adding glutaraldehyde to ensure that the concentration of glutaraldehyde is 0.01wt%, stirring for 30min at 4 ℃, adjusting the pH to 7.5-8.0 by using sodium hydroxide solution, pouring the defoamed solution into a tray according to 0.3-0.5 g/cm 2 after defoamed treatment, and incubating for 60min at 37 ℃ to form gel;
(3) And (3) washing the gel obtained in the step (2) with PBS buffer solution for multiple times, then washing with 75% ethanol by volume fraction, finally washing with purified water, taking out the gel after washing, placing the gel on a filter paper stack, placing a 200-mesh nylon net on the gel, placing 80g of heavy objects on the nylon net, and extruding the hydrogel to obtain the upper collagen layer.
(4) Coating a layer of 0.8wt% lower collagen solution on one surface of the upper collagen layer obtained in the step (3), then freezing in an ultralow temperature refrigerator at-60 ℃, transferring to a freeze dryer for freeze drying, and screening (linear mesh) the collagen film by a screening machine to obtain the double-layer collagen film.
Comparative example 1
The comparative example provides a single-layer collagen film which is an uncrosslinked collagen layer, and the preparation method comprises the following steps:
(1) Preparing a 0.5wt% collagen solution (the same amount and type as the lower collagen solutions of examples 1 and 2);
(2) And (3) placing the collagen solution in an ultralow temperature refrigerator at the temperature of minus 60 ℃ for freezing, transferring the collagen solution to a freeze dryer for freeze drying, and carrying out high-temperature treatment at the temperature of 105 ℃ for 24 hours under vacuum to obtain the single-layer collagen film.
Comparative example 2
The comparative example provides a single-layer collagen film, the preparation method of which comprises the following steps:
(1) Preparing a 0.8wt% collagen solution (the same amount and type as the lower collagen solution of example 3);
(2) And (3) placing the collagen solution in an ultralow temperature refrigerator at the temperature of minus 60 ℃ for freezing, transferring the collagen solution to a freeze dryer for freeze drying, and carrying out high-temperature treatment at the temperature of 105 ℃ for 24 hours under vacuum to obtain the single-layer collagen film.
Comparative example 3
This comparative example provides a bilayer collagen membrane differing from example 1 only in that the lower collagen layer is a crosslinked collagen layer, and in the preparation method, glutaraldehyde is added to the lower collagen solution so that the concentration of glutaraldehyde is 0.01wt%, followed by stirring for 60 minutes; other raw materials, amounts and preparation methods were the same as in example 1.
The results of the measurements of the thickness, density, porosity, mesh length and pitch of the collagen films provided in examples 1 to 3 and comparative examples 1 to 3 of the present invention are shown in Table 1.
The thickness test method comprises the following steps: the test was performed with a vernier caliper (taking care not to crush the sample too hard), 3 replicates each, and the thickness was averaged.
The apparent density test method comprises the following steps: each sample was cut into 3 pieces of 3cm x 2cm gauge, the thickness was measured with a vernier caliper, and the measured values were recorded, and the density was calculated in mg/cm 3 according to the formula ρ=m/v, and the average value was taken, expressed as mean ± standard deviation.
Porosity: each sample was cut into 3 pieces of 3cm x 2cm specification, and dried in a vacuum oven, after the drying was completed, the porosity was measured separately by the vacuum method in GB/T1966-1996, and the sample was completely submerged without air bubbles on the sample when the measurement was performed. The calculation formula is as follows: porosity= (M 1-M0)/(M1-M2) ×100% formula: m 0 = dry weight of sample, g; m 1 = weight of saturated sample in air, g; m 2 = weight of saturated sample in water, g. The porosity was averaged and expressed as mean ± standard deviation.
Mesh length and spacing: each sample was tiled with the collagen membrane top layer facing up, 10 meshes of each sample were randomly measured with vernier calipers, and then averaged, expressed as mean ± standard deviation.
Wherein the thickness and apparent density test samples are an upper collagen layer and a lower collagen layer; the testing method of the upper collagen layer comprises the following steps: directly freeze-drying the obtained upper collagen layer without coating the lower collagen solution, and then testing; the test method of the lower collagen layer comprises the following steps: the lower collagen solution was directly freeze-dried and tested. Porosity and mesh length spacing, the test sample was a collagen membrane.
TABLE 1
"/" Indicates no test is required, or that the test is meaningless.
Test example 1
The double-layer collagen films provided in examples 1 to 3 and comparative example 3 and the single-layer collagen films provided in comparative examples 1 and 2 were tested for tensile strength; the testing method comprises the following steps: each sample was cut into 5 pieces of 4cm×1cm specification, and its thickness was measured with a vernier caliper in the range of test length, the thickness was taken as a median, 5 parallel samples, and the tensile strength of the test results was expressed as mean ± standard deviation, and the test results are shown in table 2.
TABLE 2
As shown in Table 2, the double-layer collagen film provided by the invention has good mechanical strength, the tensile strength is 0.3-0.35 MPa, and the strength is far higher than that of a single-layer collagen film, and can provide good mechanical support when being used for tissue filling.
Test example 2
The double-layer collagen films provided in examples 1 to 3 were tested for cytotoxicity; the testing method comprises the following steps: the leaching ratio of each sample was 3cm 2/mL, part 5 of the medical device biological evaluation according to GB/T16886.5-2017: in vitro cytotoxicity assays were performed, 3 replicates, averaged and expressed as mean ± standard deviation. The test results are shown in Table 3.
TABLE 3 Table 3
| Test sample | Cell viability (%) |
| Example 1 | 99.43±0.83 |
| Example 2 | 99.93±0.84 |
| Example 3 | 98.77±0.85 |
As shown in Table 3, the double-layer collagen membrane provided by the invention has good biocompatibility, and the cell survival rate reaches 98% or more.
Test example 3
Taking the double-layer collagen films provided in example 1 and comparative example 3 and the single-layer collagen film provided in comparative example 1 as examples, the in vitro degradation performance of the collagen films was tested; the testing method comprises the following steps: cutting the sample of example 1, the sample of comparative example 3 and the sample of comparative example 1 to 3cm×2cm, placing the cut samples in a culture dish, respectively marking the cut samples as test 1 group, test 2 group and test 3 group, preparing collagenase (Sigma, from Clostridium histolyticum, product number: C0130, CAS: 9001-12-1) solution with a concentration of 3.5U/mL by using a tris (hydroxymethyl) aminomethane-HCl buffer (pH 7.4), adding the solution according to a proportion of 2.4mL/cm 2, placing the solution in a shaking table at 37 ℃ for shaking degradation at 50rpm, and recording the degradation process; the degradation result of the double-layer collagen film provided in example 1 is shown in fig. 2, wherein fig. 2-1 is an effect graph of degradation for 0 h; FIG. 2-2 is a graph showing the effect of degradation for 7 h; FIGS. 2-3 are graphs showing the effect of degradation for 11 h; FIGS. 2-4 are graphs showing the effect of degradation 22 h.
The degradation result of the single-layer collagen film provided in comparative example 1 is shown in fig. 3, wherein fig. 3-1 is an effect graph of degradation for 0 h; FIG. 3-2 is a graph showing the effect of degradation for 7 h; FIG. 3-3 is a graph showing the effect of degradation for 11 h; fig. 3-4 are graphs showing the effect of degradation 22 h.
The degradation results of the double-layered collagen membrane provided in comparative example 3 are shown in fig. 4; wherein FIG. 4-1 is an effect graph of degradation for 0 h; FIG. 4-2 is a graph showing the effect of degradation for 7 h; FIG. 4-3 is a graph showing the effect of degradation for 11 h; fig. 4-4 are graphs showing the effect of degradation 22 h.
As can be seen from fig. 2 to 4, the degradation rate of the lower layer of the double-layer collagen film provided by the invention is high, most of the degradation starts at 7 hours, and 22 hours are completely degraded, and only the upper collagen layer is left; the monolayer collagen film provided in comparative example 1 was mostly degraded at 7 hours, and was completely degraded for 22 hours; in contrast, the double-crosslinked double-layer collagen film provided in comparative example 3 has no degradation of the lower collagen layer (crosslinked) basically in the degradation process, has a slow degradation rate, cannot achieve the effect of rapid degradation, and cannot provide nutrition to the wound surface in time.
Test example 4
The morphology of the bilayer collagen films provided in examples 1-3 was characterized using Scanning Electron Microscopy (SEM).
Wherein, the morphology of the bilayer collagen membrane provided in example 1 is shown in fig. 5; FIG. 5-1 is an SEM image of the upper collagen layer of the double-layered collagen film provided in example 1 when the double-layered collagen film is not net-laid; FIG. 5-2 is an SEM image of the collagen layer of the double-layered collagen film provided in example 1 without screening; FIGS. 5-3 are SEM images of a bilayer collagen membrane provided in example 1 (as can be seen from the figures, the collagen membrane contains vertically-extending channels); fig. 5-4 are side SEM images of the bilayer collagen membrane provided in example 1.
The morphology of the bilayer collagen membrane provided in example 2 is shown in fig. 6; FIG. 6-1 is an SEM image of the upper collagen layer of the double-layered collagen film provided in example 2 when the double-layered collagen film is not net-laid; fig. 6-2 is an SEM image of the collagen layer of the double-layered collagen film provided in example 2 without screening.
The side morphology of the bilayer collagen membrane provided in example 3 is shown in fig. 7.
The applicant declares that the above is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be apparent to those skilled in the art that any changes or substitutions that are easily conceivable within the technical scope of the present invention disclosed by the present invention fall within the scope of the present invention and the disclosure.
Claims (10)
1. A double-layer collagen membrane, characterized in that the double-layer collagen membrane comprises an upper collagen layer and a lower collagen layer which are sequentially laminated;
The upper collagen layer is a crosslinked collagen layer; the lower collagen layer is an uncrosslinked collagen layer;
the double-layer collagen membrane has a mesh structure which is penetrated up and down.
2. The bilayer collagen membrane according to claim 1, wherein the upper collagen layer has an apparent density of 35 to 120mg/cm 3;
Preferably, the thickness of the upper collagen layer is 0.5-1.5 mm;
Preferably, the upper collagen layer is loaded with an antibacterial drug;
preferably, the antibacterial agent comprises any one or a combination of at least two of tannic acid, polyphosphate, polyhexamethylene biguanide hydrochloride, polypeptide or bletilla striata polysaccharide.
3. The bilayer collagen membrane according to claim 1 or 2, wherein the lower collagen layer has an apparent density of 15-80 mg/cm 3;
preferably, the thickness of the lower collagen layer is 0.3 to 1.5mm.
4. A bilayer collagen membrane according to any one of claims 1 to 3 wherein the mesh structure comprises at least one of a linear mesh, a diamond mesh or a circular mesh;
preferably, the length of the linear mesh is 1-5 mm;
Preferably, the areas of the diamond-shaped meshes and the round meshes are respectively and independently 0.03-3.20 mm 2;
Preferably, the distance between the two adjacent rows of meshes is 0.2-2 mm.
5. The bilayer collagen membrane according to any one of claims 1 to 4 wherein the bilayer collagen membrane has a porosity of greater than or equal to 75%;
Preferably, the materials of the upper and lower collagen layers each independently comprise any one or a combination of at least two of type I collagen, type II collagen, or type III collagen;
preferably, the degradation time of the lower collagen layer is less than or equal to 24 hours.
6. A method of producing a bilayer collagen membrane according to any one of claims 1 to 5, comprising the steps of:
And laminating the upper collagen layer and the lower collagen layer, and preparing meshes to obtain the double-layer collagen film.
7. The method of claim 6, wherein the method of preparing the upper collagen layer comprises the steps of:
mixing the upper collagen solution, phosphate buffer solution and cross-linking agent, regulating the pH value of the system to 7.5-8 after cross-linking, and then incubating to obtain gel; washing and extruding the gel to obtain the upper collagen layer;
Preferably, the weight fraction of the collagen in the upper collagen solution is 0.3-0.8%;
Preferably, the upper collagen solution further comprises an antibacterial agent;
preferably, the mass ratio of the upper collagen solution to the phosphate buffer is (8-10): 1.
8. The method of claim 7, wherein the cross-linking agent is glutaraldehyde;
preferably, the mass concentration of the cross-linking agent in the system is 0.01-0.1%;
preferably, the temperature of the crosslinking is 0-10 ℃ and the time is 0.5-3 h;
preferably, the temperature of the incubation is 35-38 ℃ and the time is 40-100 min.
9. The method of preparing a lower collagen layer according to claim 7 or 8, comprising the steps of:
Coating a lower collagen solution on one surface of the upper collagen layer, and freeze-drying to obtain a laminated upper collagen layer and lower collagen layer;
preferably, the weight fraction of the collagen in the lower collagen solution is 0.3-0.8%;
Preferably, the apparatus for preparing a mesh comprises a netmaker and/or a laser.
10. A dermis restoration material, characterized in that it comprises a double-layer collagen film according to any one of claims 1 to 5.
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