CN111714695B - Density double-layer biological membrane, preparation method and application - Google Patents
Density double-layer biological membrane, preparation method and application Download PDFInfo
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Abstract
The invention provides a preparation method of a density double-layer biomembrane, S1, preparing a natural polymer biomedical material into a solution with the concentration of 5-10 wt% as a dense layer solution; preparing a natural polymer biomedical material into a 1.5-2.5 wt% solution as a loose layer solution; respectively centrifuging the dense layer solution and the loose layer solution for 5-10 minutes by using a refrigerated centrifuge for defoaming; s2, pouring the dense layer solution into a custom-made appliance, vibrating and leveling, air-drying at the temperature of 5-15 ℃ and the humidity of 30-70% until the concentration of the dense layer solution is 20-80 wt%, and then freezing at-20 ℃ to-40 ℃ for 1-2 hours to form a dense layer; s3, pouring the loose layer solution onto the frozen compact layer obtained in the step S3, vibrating and flattening, and unfreezing at 1-4 ℃; after the compact layer is melted, putting the compact layer into a refrigerated centrifuge for centrifuging for 5-10 minutes at the temperature of 6-7 ℃ and at the rotating speed of 3200-3500 rpm; s4, after vacuum freeze drying, heating at 50-70 ℃ for 1-2 hours for fixation, and irradiating by adopting electron rays with the irradiation dose of 10-30 kGy. The two layers of the film are tightly bonded.
Description
Technical Field
The invention relates to a density double-layer biomembrane, a preparation method and application thereof, belonging to the technical field of medical appliances.
Background
The dense-dense double-layer biomembrane is a repairing material frequently used in clinic. The loose structure on one side can promote the repair and regeneration of tissue disease defects, and the compact layer on one side can effectively prevent the growth of fibroblasts.
However, the current widely used bilayer membrane in clinic still has some defects in product performance.
Firstly, the double-layer membrane often has the phenomenon of two-layer membrane separation in the process of normal saline soaking, and the operation and the use effect are influenced. The reason is presumed that the existing method for preparing the dense-dense double-layer biological membrane only presses two layers of membranes which are respectively lyophilized together, and the two layers of membranes have no mutual acting force basically, so the membranes are easy to separate. Chinese patent CN107412869A is prepared by adhering two films of fibrinogen and prothrombin solution, which increases the cost and complexity of operation. In addition, the addition of other substances has unknown influence on the safety and effectiveness of the product.
In addition, due to the limitation of the configuration concentration of the polymer solution, the freeze-dried dense layer still has larger pore diameter and porosity, can not effectively prevent cells from growing in, has insufficient barrier capability of the dense layer membrane, can not provide continuous space for guiding tissue regeneration for the loose layer and the like, and leads to the unsatisfactory tissue repair function of the membrane.
Disclosure of Invention
The invention provides a density double-layer biological membrane and a preparation method thereof, which can effectively solve the problems.
The invention is realized in the following way:
a preparation method of a density double-layer biological membrane comprises the following steps:
s1, preparing a 5-10 wt% solution of the natural polymer biomedical material as a dense layer solution; preparing a natural polymer biomedical material into a 1.5-2.5 wt% solution as a loose layer solution; respectively centrifuging the dense layer solution and the loose layer solution for 5-10 minutes by using a refrigerated centrifuge at the temperature of 3-5 ℃ and the rotating speed of 2800-3200 rpm for defoaming;
s2, pouring the dense layer solution into a custom-made appliance, vibrating and leveling, air-drying at the temperature of 5-15 ℃ and the humidity of 30-70% until the concentration of the dense layer solution is 20-80 wt%, and then freezing at-20 ℃ to-40 ℃ for 1-2 hours to form a dense layer;
s3, pouring the loose layer solution onto the frozen compact layer obtained in the step S3, vibrating and flattening, and unfreezing at 1-4 ℃; after the compact layer is melted, putting the compact layer into a refrigerated centrifuge for centrifuging for 5-10 minutes at the temperature of 6-7 ℃ and at the rotating speed of 3200-3500 rpm;
s4, after vacuum freeze drying, heating at 50-70 ℃ for 1-2 hours for fixation, pressing into the required thickness, and irradiating by adopting electron rays with the irradiation dose of 10-30 kGy;
S5, cross-linking with aldehyde cross-linking agent, and vacuum freeze drying to obtain the dense-dense double-layer biomembrane.
As a further improvement, the natural polymer biomedical material is one or more of chitosan, hyaluronic acid, sodium alginate and collagen.
As a further improvement, the air-drying air-dries the dense layer to a dense layer solution concentration of 40-80 wt%.
The further improvement is that the vacuum freeze drying is carried out under the condition that the vacuum degree is 80-100 millitorr, the temperature is kept for 30min at the temperature of 2-3 ℃, the temperature is kept for 80-110 min at the temperature of-35-25 ℃, the temperature is kept for 12-14 h at the temperature of 0 ℃, and the temperature is kept for 12-16 h at the temperature of 10-15 ℃.
As a further improvement, after the step S4, 3-5 wt% ammonia water solution is used for neutralization for 12-24 hours.
As a further improvement, washing to neutrality is also carried out after neutralization with an aqueous ammonia solution.
The invention also provides a density double-layer biological membrane prepared by the method.
The invention also provides application of the density double-layer biomembrane in preparation of a tissue repair material.
The invention has the beneficial effects that:
the dense layer is air-dried until the concentration of the solution is 20-80 wt%, so that the density of the dense layer can be greatly increased, fibroblast is effectively prevented from growing and proliferating, a continuous tissue regeneration guiding space is provided for the loose layer, and the tissue repair effect is improved.
After the dense layer solution is frozen, the loose layer is dissolved and poured into the upper layer, then unfreezing is carried out, the dense layer is waited for melting, and then centrifugal operation is carried out, so that the solution is directly contacted and fused to increase the adhesion of the two layers, the tightness of the two layers is further increased by heating solidification and electron radiation irradiation, and the phenomenon of membrane separation of the two layers cannot occur in the process of soaking in normal saline.
The invention does not need to add a binder for bonding, saves the cost and ensures the safety of the product.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a schematic diagram of a dense-dense double-layer biofilm provided in example 1 of the present invention.
FIG. 2 is a graph showing the results of the cell penetration test in example 11 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings of the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Example 1
A preparation method of a density double-layer biological membrane comprises the following steps:
s1, preparing 8 wt% solution of chitosan as dense layer solution; preparing 2 wt% solution of chitosan as loose layer solution; the pH value of the dense layer solution is 3.4, and the pH value of the loose layer solution is 4.5; respectively centrifuging the dense layer solution and the loose layer solution for 10 minutes by using a refrigerated centrifuge at the temperature of 4 ℃ and the rotating speed of 3200 rpm for defoaming;
s2, pouring the dense layer solution into a custom-made appliance, vibrating and leveling, air-drying at the temperature of 10 ℃ and the humidity of 50% until the concentration of the dense layer solution is 60 wt%, and then freezing at-30 ℃ for 2 hours to form a dense layer;
s3, pouring the loose layer solution onto the frozen compact layer obtained in the step S3, vibrating and flattening, and unfreezing at 2 ℃; after the compact layer is melted, putting the compact layer into a refrigerated centrifuge for centrifuging for 8 minutes at the temperature of 6 ℃ and the rotating speed of 3500 rpm to ensure that the two layers are more compact;
s4, after vacuum freeze drying, heating at 60 ℃ for 1 hour for fixing, pressing to a required thickness, and irradiating by adopting electron rays with 30kGy irradiation dose; neutralizing with 4 wt% ammonia solution for 18 hr, and washing to neutrality; the vacuum freeze drying is to keep the temperature at 2 ℃ for 30min, the temperature at-35 ℃ for 110min, the temperature at 0 ℃ for 12h and the temperature at 10 ℃ for 12h under the condition that the vacuum degree is 80 mTorr;
And S5, cross-linking with aldehyde cross-linking agent, cleaning to neutrality, and vacuum freeze-drying to obtain the dense-dense double-layer biomembrane. The vacuum freeze drying is performed under the condition that the vacuum degree is 100 mTorr, the temperature is kept at 3 ℃ for 30min, the temperature is kept at-25 ℃ for 80min, the temperature is kept at 0 ℃ for 14h, and the temperature is kept at 15 ℃ for 16 h.
The prepared dense-dense double-layer biomembrane can not generate the phenomenon of separation of two layers of membranes after being soaked in normal saline for 24 hours.
After the film was immersed in water for 30 minutes, the peel strength between the two films was measured to be 2.15N/cm using a 180 ℃ peel strength tester.
Example 2
A preparation method of a density double-layer biological membrane comprises the following steps:
s1, preparing hyaluronic acid into a solution with the weight percent of 10% as a dense layer solution; preparing hyaluronic acid into a 2.5 wt% solution as a loose layer solution; the pH value of the dense layer solution is 3.8, and the pH value of the loose layer solution is 5.0; respectively centrifuging the dense layer solution and the loose layer solution for 8 minutes by using a refrigerated centrifuge at the temperature of 3 ℃ and the rotating speed of 3200 rpm for defoaming;
s2, pouring the dense layer solution into a custom-made appliance, vibrating and leveling, air-drying at the temperature of 8 ℃ and the humidity of 40% until the concentration of the dense layer solution is 50 wt%, and then freezing at-20 ℃ for 1.5 hours to form a dense layer;
S3, pouring the loose layer solution onto the frozen compact layer obtained in the step S3, vibrating and flattening, and unfreezing at 4 ℃; after the compact layer is melted, putting the compact layer into a refrigerated centrifuge for centrifuging for 10 minutes at the temperature of 7 ℃ and the rotating speed of 3500 rpm to ensure that the two layers are more compact;
s4, after vacuum freeze drying, heating at 50 ℃ for 2 hours for fixation, pressing to a required thickness, and irradiating by adopting electron rays with the irradiation dose of 20 kGy; neutralizing with 4 wt% ammonia solution for 18 hr, and washing to neutrality; the vacuum freeze drying is performed under the condition that the vacuum degree is 100 mTorr, the temperature is kept at 2 ℃ for 30min, the temperature is kept at-25 ℃ for 100min, the temperature is kept at 0 ℃ for 14h, and the temperature is kept at 10 ℃ for 14 h.
And S5, cross-linking with aldehyde cross-linking agent, cleaning to neutrality, and vacuum freeze-drying to obtain the dense-dense double-layer biomembrane. The vacuum freeze drying is performed under the condition that the vacuum degree is 100 mTorr, the temperature is kept at 3 ℃ for 30min, the temperature is kept at-25 ℃ for 80min, the temperature is kept at 0 ℃ for 14h, and the temperature is kept at 15 ℃ for 16 h.
The prepared dense-dense double-layer biomembrane can not generate the phenomenon of separation of two layers of membranes after being soaked in normal saline for 24 hours.
After the film was immersed in water for 30 minutes, the peel strength between the two films was measured to be 0.98N/cm using a 180 ℃ peel strength tester.
Example 3
A preparation method of a density double-layer biological membrane comprises the following steps:
s1, preparing 8 wt% solution of sodium alginate as dense layer solution; preparing sodium alginate into a 2 wt% solution as a loose layer solution; the pH value of the dense layer solution is 3.2, and the pH value of the loose layer solution is 4.0; respectively centrifuging the dense layer solution and the loose layer solution for 10 minutes by using a refrigerated centrifuge at the temperature of 4 ℃ and the rotating speed of 3200 rpm for defoaming;
s2, pouring the dense layer solution into a custom-made appliance, vibrating and leveling, air-drying at the temperature of 15 ℃ and the humidity of 70% until the concentration of the dense layer solution is 60 wt%, and then freezing at-30 ℃ for 1 hour to form a dense layer;
s3, pouring the loose layer solution onto the frozen compact layer obtained in the step S3, vibrating and flattening, and unfreezing at 2 ℃; after the compact layer is melted, putting the compact layer into a refrigerated centrifuge for centrifuging for 8 minutes at the temperature of 6 ℃ and the rotating speed of 3500 rpm to ensure that the two layers are more compact;
s4, after vacuum freeze drying, heating at 70 ℃ for 1 hour for fixing, pressing to the required thickness, and irradiating by adopting electron rays with the irradiation dose of 10 kGy; the vacuum freeze drying is performed under the condition that the vacuum degree is 80 mTorr, the temperature is kept at 2 ℃ for 30min, the temperature is kept at 35 ℃ for 110min, the temperature is kept at 0 ℃ for 12h, and the temperature is kept at 10 ℃ for 12 h.
S5, cross-linking with aldehyde cross-linking agent, and vacuum freeze drying to obtain the dense-dense double-layer biomembrane. The vacuum freeze drying is performed under the condition that the vacuum degree is 100 mTorr, the temperature is kept at 3 ℃ for 30min, the temperature is kept at 25 ℃ for 80min, the temperature is kept at 0 ℃ for 14h, and the temperature is kept at 15 ℃ for 16 h.
The prepared dense-dense double-layer biomembrane can not generate the phenomenon of separating two layers of membranes after being soaked in normal saline for 24 hours.
After the film was immersed in water for 30 minutes, the peel strength between the two films was measured to be 1.33N/cm using a 180 ℃ peel strength tester.
Example 4
A preparation method of a density double-layer biological membrane comprises the following steps:
s1, preparing collagen into a solution with the concentration of 10 wt% as a dense layer solution; preparing 2.5 wt% collagen solution as loose layer solution; the pH value of the dense layer solution is 3.2, and the pH value of the loose layer solution is 4.0; respectively centrifuging the dense layer solution and the loose layer solution for 10 minutes by using a refrigerated centrifuge at the temperature of 4 ℃ and the rotating speed of 3200 rpm for defoaming;
s2, pouring the dense layer solution into a custom-made appliance, vibrating and leveling, air-drying at the temperature of 15 ℃ and the humidity of 70% until the concentration of the dense layer solution is 60 wt%, and then freezing at-30 ℃ for 1 hour to form a dense layer;
S3, pouring the loose layer solution onto the frozen compact layer obtained in the step S3, vibrating and flattening, and unfreezing at 2 ℃; after the compact layer is melted, putting the compact layer into a refrigerated centrifuge for centrifuging for 8 minutes at the temperature of 6 ℃ and the rotating speed of 3500 rpm to ensure that the two layers are more compact;
s4, after vacuum freeze drying, heating at 60 ℃ for 1 hour for fixing, pressing to a required thickness, and irradiating by adopting electron rays with the irradiation dose of 20 kGy; the vacuum freeze drying is carried out under the condition that the vacuum degree is 80 mTorr, the temperature is kept at 2 ℃ for 30min, the temperature is kept at 35 ℃ for 110min, the temperature is kept at 0 ℃ for 12h, and the temperature is kept at 10 ℃ for 12 h.
And S5, cross-linking with an aldehyde cross-linking agent, and performing vacuum freeze drying to obtain the dense-dense double-layer biomembrane. The vacuum freeze drying is performed under the condition that the vacuum degree is 100 mTorr, the temperature is kept at 3 ℃ for 30min, the temperature is kept at-25 ℃ for 80min, the temperature is kept at 0 ℃ for 14h, and the temperature is kept at 15 ℃ for 16 h.
The prepared dense-dense double-layer biomembrane can not generate the phenomenon of separation of two layers of membranes after being soaked in normal saline for 24 hours.
After the film was immersed in water for 30 minutes, the peel strength between the two films was measured to be 1.28N/cm using a 180 ℃ peel strength tester.
Example 5
Without electron irradiation, the peel strength of the two layers was measured to be 1.52N/cm as in example 1.
Example 6
The two layers were tested to have a peel strength of 0.16N/cm as in example 1 without thawing and thawing.
Example 7
After melting, the two layers were tested for peel strength of 0.87N/cm in the same manner as in example 1 without centrifugation.
Example 8
The two layers were tested to have a peel strength of 0.08N/cm as in example 2 without thawing and thawing.
Example 9
The two layers were tested for peel strength of 0.13N/cm as in example 3 without thawing and thawing.
Example 10
After melting, the two layers were tested for peel strength of 0.10N/cm as in example 4 without centrifugation.
Example 11
The dense layer solution was 5% chitosan, and was not air dried, and other samples were made in the same manner as example 1, and the peel strength of the two layers was 1.92N/cm.
Example 12 cell penetration assay
Preparing a 5% dense layer and a 2.5 wt% loose layer by using collagen, air-drying the solution until the concentration of the solution is 20%, 40%, 60% and 80%, adding the loose layer without melting, and directly freeze-drying (facilitating double-layer membrane wind power), wherein other operation methods are the same as those of example 4, so that a plurality of double-layer membranes with different densities of the dense layers are prepared.
And (3) putting the double-layer membrane compact layer upwards into a cell culture dish, and dripping cell culture solution to completely infiltrate the membrane. Preparing L929 cell suspension, carefully inoculating 5000 cells on the surface of the compact layer, and adding cell culture solution for culture after the cells are attached to the membrane. After 72 hours (at this time, the cells did not proliferate out of the boundary of the membrane), the membrane was carefully removed, the double-layer membrane was carefully peeled off with forceps, the dense layer was discarded, the loose layer was placed in a trypsin solution to digest the cells, then the cells were repeatedly washed with a cell culture solution, the trypsin solution and the washing solution were combined and centrifuged, the supernatant was discarded, the cells were re-seeded in a 24-well cell culture plate, and after culturing for 48 hours, the cell proliferation was measured by the CCK8 method. The experimental result can reflect the cell retardation capacity of the compact layer membrane. The results of the experiment are shown in FIG. 2.
As shown in FIG. 2, the proliferation rate of cells gradually decreased with the increase of the concentration of the dense layer solution, and when the concentration was increased to 60%, the relative proliferation rate decreased to about one tenth of that of 5%, and when the concentration was increased to 60%, the relative proliferation rate did not substantially decrease any more. The result shows that when the concentration is 60%, the compact layer has large blocking capacity on cells and plays a better barrier role.
Example 13 use Effect test of bilayer film prepared by the present invention
6 adult Beagle dogs are selected to establish a mandibular posterior dental area periodontal defect model. The mandibular premolars 2 and 3 of each dog were randomized into 4 groups of experimental teeth (24 total): blank control group, experimental group one, experimental group two, experimental group three, 6 teeth per group. Animal anesthesia and fixation, disinfection, drape, cheek bone exposure, and alveolar bone removal by using a split drill and an osteotome, wherein the bone defect range comprises: the vertical height is about 4mm, and the horizontal height is about 3 mm. The membrane material is cut into a proper shape, the edge of the membrane covers 2-3mm beyond the periphery of the bone defect, the upper end of the membrane is slightly lower than the gingival margin, the gingival flap is reset, and the membrane is intermittently sutured. The blank control group was not directly sutured using a film, the first experimental group was a double-layer film prepared in example 1, the second experimental group was a double-layer film prepared in example 11, and the third experimental group was a collagen film product for general medical use. Animals were sacrificed 12 weeks after surgery, the jaw bone was removed to prepare tissue sections, and the height of the new alveolar bone was measured by observation under a light microscope. The observation results are shown in table 1.
TABLE 1
As can be seen from table 1, the first experimental group showed more significant increase in new alveolar bone than the blank control group, the second experimental group, and the third experimental group.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. A preparation method of a density double-layer biological membrane is characterized by comprising the following steps:
s1, preparing a 5-10 wt% solution of the natural polymer biomedical material as a dense layer solution; preparing a natural polymer biomedical material into a 1.5-2.5 wt% solution as a loose layer solution; respectively centrifuging the dense layer solution and the loose layer solution for 5-10 minutes by using a refrigerated centrifuge at the temperature of 3-5 ℃ and the rotating speed of 2800-3200 rpm for defoaming;
s2, pouring the dense layer solution into a custom-made appliance, vibrating and leveling, air-drying at the temperature of 5-15 ℃ and the humidity of 30-70% until the concentration of the dense layer solution is 40-80 wt%, and then freezing at-20 ℃ to-40 ℃ for 1-2 hours to form a dense layer;
S3, pouring the loose layer solution onto the frozen compact layer obtained in the step S2, vibrating and flattening, and unfreezing at 1-4 ℃; after the compact layer is melted, putting the compact layer into a refrigerated centrifuge for centrifuging for 5-10 minutes at the temperature of 6-7 ℃ and at the rotating speed of 3200-3500 rpm;
s4, after vacuum freeze drying, heating at 50-70 ℃ for 1-2 hours for fixation, pressing into the required thickness, and irradiating by adopting electron rays with the irradiation dose of 10-30 kGy;
s5, cross-linking with an aldehyde cross-linking agent, and then carrying out vacuum freeze drying to obtain the density double-layer biomembrane;
and neutralizing for 12-24 hours by using 3-5 wt% ammonia water solution after the step S4.
2. The method for preparing a dense-dense double-layer biomembrane as claimed in claim 1, wherein the natural polymer biomedical material is one or more of chitosan, hyaluronic acid, sodium alginate and collagen.
3. The method for preparing a dense-dense double-layer biofilm according to claim 1, wherein the vacuum freeze drying is performed under a vacuum degree of 80 to 100 mTorr, the temperature is kept at 2 to 3 ℃ for 30min, the temperature is kept at-35 to-25 ℃ for 80 to 110min, the temperature is kept at 0 ℃ for 12 to 14h, and the temperature is kept at 10 to 15 ℃ for 12 to 16 h.
4. The method for preparing a dense-dense double-layer biofilm according to claim 1, wherein the biofilm is further washed to be neutral after being neutralized by an ammonia solution.
5. A dense-dense bilayer biofilm prepared by the method of claims 1-4.
6. Use of the dense-dense double-layer biofilm of claim 5 in the preparation of a tissue repair material.
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