CN117797334A - Guided tissue regeneration membrane and preparation method thereof - Google Patents
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- Materials For Medical Uses (AREA)
Abstract
The invention provides a preparation method of a guided tissue regeneration membrane, which comprises the following steps: (1) repeatedly freezing and thawing after removing the extra impurities of the pericardium; (2) performing decellularization treatment on the pericardium in the step (1); (3) cleaning the pericardium of step (2) with a cleaning agent; (4) treating the pericardium of step (3) with citric acid; (5) Immersing the pericardium of step (4) in a solution containing a short peptide; (6) lyophilizing the pericardium of step (5). The guided tissue regeneration membrane prepared by the method has a compact layer and loose layer double-layer structure, can better guide tissue regeneration and has more excellent clinical use effect; the guided tissue regeneration membrane has the advantages of mild preparation process, cell removal and reservation of beneficial components such as various growth factors.
Description
Technical Field
The invention relates to the technical field of biological materials, in particular to a tissue regeneration guiding membrane and a preparation method thereof.
Background
The concept of Guiding Tissue Regeneration (GTR) was first proposed by N yman, etc., which refers to selectively guiding cell attachment proliferation to a damaged site by means of mechanical barrier, etc., to achieve the purpose of tissue repair. The key to realizing tissue repair and regeneration is the development and application of functional repair materials. Guided tissue regeneration membranes for clinical applications can be classified into non-degradable and degradable absorption membranes according to raw materials. The non-degradable absorbing GTR film mainly comprises a titanium film and an expanded polytetrafluoroethylene (e-PTFE) film, and has more defects: the membrane is not easy to fix; the complications such as mucous membrane rupture, membrane exposure, infection and the like are easy to occur; the secondary operation is needed to remove, the damage to the tissues is easy to cause, and the effect of continuous osteogenesis is affected. After the idea of biodegradation occurs at the end of the 80 th century, a degradable absorption film material is introduced, so that the treatment time is shortened, the biocompatibility is better than that of an artificially synthesized high polymer, and the biodegradable absorption film has become a general development trend for guiding a tissue regeneration film technology.
Currently, there are few products of degradable and absorbable guided tissue regeneration membrane materials on the market, and there are some drawbacks. The current GTR membrane prepared by collagen has the following defects: the mechanical property is poor, the structure is easy to collapse when meeting water, the degradation is too fast, and the growth speed of the bone tissue is not matched; and insufficient porosity results in limited cell growth space, which is unfavorable for bone tissue growth, resulting in slow window healing. Therefore, seeking a guided tissue regeneration membrane with good mechanical properties and high porosity is a technical problem to be solved urgently in the field.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a guided tissue regeneration membrane and a preparation method thereof. The guided tissue regeneration membrane prepared by the invention has excellent mechanical property, good biocompatibility and low immune response, and can be produced in a large scale, and the degradation speed of the guided tissue regeneration membrane is matched with the growth speed of bone tissue.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method for preparing a guided tissue regeneration membrane, comprising the steps of:
(1) Repeatedly freezing and thawing after removing the redundant impurities of the pericardium;
(2) Performing decellularization treatment on the pericardium in the step (1);
(3) Cleaning the pericardium of the step (2) by using a cleaning agent;
(4) Treating the pericardium of step (3) with citric acid;
(5) Immersing the pericardium of step (4) in a solution containing a short peptide;
(6) And (5) freeze-drying the pericardium in the step (5).
Preferably, the pericardium of step (1) comprises bovine pericardium, porcine pericardium or ovine pericardium.
Preferably, the repeated freeze thawing specific operation of the step (1) is as follows: and (3) placing the pericardium after removing the redundant impurities in PBS liquid, freezing for 2-4 hours at the temperature of-80 ℃ to-50 ℃, then taking out the pericardium and cleaning in a water bath for 30-60min, and repeatedly freezing and thawing for 2-4 times.
Preferably, before said step (2), said sterilizing the pericardium of step (1) is further included; the specific operation of the disinfection is as follows: taking out pericardium, immersing in 75% (v/v) alcohol for 2-4 hr, and changing liquid once; immersing the processed pericardium in PBS liquid, placing in a shaking table, and cleaning at a speed of 60-120 rpm for 4-6h, and changing the PBS liquid every 2h.
Preferably, the step (2) decellularizing treatment specific operation is: putting the treated pericardium into pancreatin solution with mass-volume percentage concentration of 0.2% -0.5% for cell removal treatment for 12-24h, and rinsing with purified water after treatment.
Preferably, the step (3) cleaning treatment specifically operates as: immersing the pericardium in a cleaning agent, and oscillating for 8-16h at a speed of 60-120 rpm in a normal temperature shaking table; the cleaning agent in the step is 0.1% -0.8% (v/v) of fatty alcohol-polyoxyethylene ether solution.
Preferably, the specific steps of the citric acid treatment in the step (4) are as follows: immersing the pericardium in 0.1-0.8M citric acid solution, oscillating at normal temperature for 0.5-2 h, and rinsing with purified water after treatment.
Preferably, the method further comprises the step of cleaning the pericardium of the step (4) before the step (5); the cleaning specific operation is as follows: taking out pericardium, immersing in PBS liquid, and placing in a shaker, and cleaning at 60-120 rpm for 8-12 hr, wherein PBS liquid is replaced every 4 hr.
Preferably, the step (5) specifically includes: the pericardium is placed in PBS solution containing 0.1-0.8 mg/mL of carbodiimide (EDC) and 0.1-1 mg/mL of succinimide (NHS) to react for 10-30 min, then washed by PBS, placed in PBS solution containing 0.1-1 mg/mL of short peptide, incubated for 2-4h at normal temperature, and rinsed by pure water to remove the redundant short peptide.
Preferably, the short peptide in the step (5) is a short peptide consisting of glycine, phenylalanine, hydroxyproline, glutamic acid, arginine (Gly-Phe-Hyp-Glu-Arg). The short peptide has certain cell adhesiveness, helps cells adhere to tissue regeneration membrane, and promotes wound healing.
Preferably, the step (6) freeze-drying is specifically performed as follows: the freeze-drying comprises a pre-freezing stage and a sublimation drying stage, wherein the pre-freezing stage comprises a first pre-freezing stage and a second pre-freezing stage, the temperature of the first pre-freezing stage is between-5 ℃ and-2 ℃, the time is between 10 and 60 minutes, and the heat preservation time is between 20 and 60 minutes; the temperature of the second pre-freezing stage is between 50 ℃ below zero and 20 ℃ below zero, the time is 20-60 min, and the heat preservation time is 40-60 min. The sublimation drying stage comprises a first sublimation drying stage, a second sublimation drying stage and a third sublimation drying stage, wherein the temperature of the first sublimation drying stage is between-5 ℃ and-2 ℃, the time is 40-120 min, the heat preservation time is 200-400 min, and the vacuum degree is set to be 20-40 Pa; the temperature of the second sublimation drying stage is 3-8 ℃, the time is 20-120 min, the heat preservation time is 20-120 min, and the vacuum degree is set to be 20-40 Pa; the temperature of the third sublimation drying stage is 10 ℃ to 20 ℃, the time is 20 to 60min, the heat preservation time is 40 to 120min, and the vacuum degree is set to be 20 to 40Pa.
According to the invention, by setting a specific freeze-drying procedure, the guided tissue regeneration collagen membrane with loose and compact layers is obtained, so that the guided tissue regeneration collagen membrane has good mechanical properties. Accordingly, the present invention provides a guided tissue regeneration membrane prepared by the above method, the guided tissue regeneration membrane comprising collagen as a component; it has a multi-layer structure including a porous layer and a dense layer.
The main components of the loose layer and the compact layer are collagen, wherein the loose layer collagen fibers are loosely arranged, and the fibers are loosely arranged; the compact layer collagen is tightly arranged, and the fibers are crossed horizontally and vertically; the special loose layer and compact layer of the pericardium of the invention show more obvious loose and compact characteristics after being treated by the process.
The invention has the beneficial effects that:
the invention prepares a guided tissue regeneration membrane by taking a pericardium as a tissue material, and carries out a series of improvements aiming at a preparation method. For example: the invention provides a novel decellularized detergent, which uses 0.1-0.8% of fatty alcohol polyoxyethylene ether solution to treat cells on pericardial tissues, and has better effect, shorter decellularized treatment time, better cell removal effect and less cell residue compared with other reagents. The porosity of the material is improved by using citric acid to treat the pericardial tissue material, the porosity can reach more than 90%, and more space for cell growth is provided. The invention also crosslinks short peptide which promotes cell adhesion on the pericardial tissue material, enhances the cell adhesion and cell aggregation of the tissue material, and ensures that cells grow along the tissue material to be beneficial to wound healing and tissue regeneration. According to the invention, a loose layer and a compact layer double-layer structure is obtained through a subsequent specific freeze-drying procedure, and the structure can improve the mechanical property of the regeneration film and accelerate the growth speed and bone density of the new bone in the bone defect area.
The guided tissue regeneration membrane prepared by the method has a compact layer and loose layer double-layer structure, and different structures have better mechanical properties and ductility, can better guide tissue regeneration and have more excellent clinical use effects; the guided tissue regeneration membrane prepared by the method has good biocompatibility and hydrophilicity, the degradation speed is matched with the bone regeneration speed, and the degradation products are harmless to human bodies; the guided tissue regeneration membrane has the advantages of mild preparation process, cell removal and reservation of beneficial components such as various growth factors.
The main component of the guided tissue regeneration membrane is collagen, and the guided tissue regeneration membrane is suitable for repairing tissue defects in the oral cavity and is also suitable for regenerating other types of tissues. The prepared guide film has the characteristics of isolating cell ingrowth and slow degradation after the densification treatment of the material, has longer retention time in vivo than the existing degradable guide film, and can provide sufficient time and space for periodontal defect growth. Can be used in combination with bone powder during surgical implantation, thereby effectively promoting regeneration of periodontal disease damaged tissues.
The preparation method is simple, the sources of raw materials are wide, the processing process is suitable for large-scale industrial production, and the cost is reduced.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
FIG. 1 is a diagram showing the comparison result of experimental photographs (3 months) of rabbit animals (FIG. A is an example, and FIG. B is a comparison example 13);
fig. 2 is a schematic diagram of histological section images comparing results.
Detailed Description
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. 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 embodiment provides a preparation method of a guided tissue regeneration membrane, wherein the pericardium used in the embodiment is a bovine pericardium, and the method comprises the following steps:
(1) Repeated freezing and thawing
And (3) placing the pericardium after removing the redundant impurities in PBS (phosphate buffer solution) and freezing for 3 hours at the temperature of minus 60 ℃, then taking out the pericardium and cleaning for 40 minutes in a water bath, and repeatedly freezing and thawing for 2-4 times. Cells were broken up by repeated freeze thawing.
(2) Disinfection
Taking out pericardium, immersing in 75% (v/v) alcohol, treating for 3 hr, and changing liquid once; immersing the processed pericardium in PBS liquid, placing in a shaking table, and cleaning at a speed of 60-120 rpm for 4-6h, and changing the PBS liquid every 2h. Harmful microorganisms on the pericardium are removed by a disinfection step.
(3) Decellularization treatment
Putting the treated pericardium into a pancreatin solution with the mass percent concentration of 0.4% for cell removal treatment for 12-24h, and rinsing with purified water after the treatment. Cells attached to the pericardium are shed by treating cells that digest the pericardium with pancreatin.
(4) Cleaning with cleaning agent
Immersing the pericardium in a cleaning agent, oscillating for 8 hours at a speed of 60-120 rpm in a normal-temperature shaking table, and rinsing with pure water; the cleaning agent is 0.5% (v/v) of fatty alcohol-polyoxyethylene ether solution. The phospholipid bilayer of the cell membrane is dissolved by the fatty alcohol polyoxyethylene ether solution, so that the purposes of removing cells and dissolving cell fragments are achieved. The invention uses a new decellularized treating agent, and can remove cells and cell residue fragments on the pericardium better than other treating agents.
Preparing fatty alcohol polyoxyethylene ether solution: mixing 5mL of fatty alcohol-polyoxyethylene ether with 1000mL of deionized water to prepare a fatty alcohol-polyoxyethylene ether solution with the volume percentage of 0.5%.
(5) Treatment with citric acid
Immersing the pericardium in citric acid solution with concentration of 0.5M, and oscillating for 0.5-2 h at normal temperature. The invention improves the porosity and increases the growth space of cells by treating the pericardium with citric acid.
(6) Cleaning
Taking out pericardium, immersing in PBS liquid, and placing in a shaker, and cleaning at 60-120 rpm for 8-12 hr, wherein PBS liquid is replaced every 4 hr.
(7) Immersing in a solution containing a short peptide;
the pericardium is placed in PBS containing 0.5mg/mL carbodiimide (EDC) and 0.5mg/mL succinimide (NHS) for reaction for 20min, washed by PBS after reaction for 10-30 min, placed in PBS containing 0.5mg/mL short peptide, incubated for 2-4h at normal temperature, and rinsed by pure water to remove the redundant short peptide.
Unlike short peptide, the protein of pericardial material has triple helix structure, so that the short peptide with adhesion effect is cross-linked to pericardial material to strengthen the adhesion effect of cell on pericardium and promote cell growth.
In this example, the short peptide used was a pentapeptide composed of glycine, phenylalanine, hydroxyproline, glutamic acid and arginine (Gly-Phe-Hyp-Glu-Arg). Gly-Phe-Hyp-Glu-Arg short peptide can be prepared by a chemical synthesis method, and is prepared into freeze-dried powder to prepare a protein solution with the concentration of 0.5 mg/mL. Experiments prove that the short peptide synthesized by the invention not only has cell adhesion effect, but also has good hydrophilicity, has good stability in a liquid environment and is not easy to degrade, the pure product with the purity of 98 percent is dissolved to prepare protein solution, and the purity of the protein solution can be maintained to be more than 90 percent after being placed for 12 months at room temperature.
(8) Freeze-drying
Lyophilizing according to the lyophilizing steps and parameters shown in table 1 to obtain guided tissue regeneration collagen membrane with loose and compact layer.
Table 1: lyophilization step and specific parameters
Example 2
Example 2 differs from example 1 in that the concentration in the fatty alcohol-polyoxyethylene ether solution is 0.1%, the concentration in the citric acid solution is 0.1M, and the concentration in the short peptide solution is 0.1mg/mL.
Example 3
Example 3 differs from example 1 in that the concentration in the fatty alcohol-polyoxyethylene ether solution is 0.8%, the concentration in the citric acid solution is 0.8M, and the concentration in the short peptide solution is 1mg/mL.
Example 4
Example 4 differs from example 1 in that the lyophilization step differs from the specific parameters, which are set forth in the following table:
table 2: lyophilization step and specific parameters
Example 5
Example 5 differs from example 1 in that the lyophilization step differs from the specific parameters, which are set forth in the following table:
table 3: lyophilization step and specific parameters
Example 6
This example differs from example 1 in that example 6 uses a porcine pericardium.
Example 7
This example differs from example 1 in that example 7 uses a sheep pericardium.
Comparative example 1
The method for preparing a guided tissue regeneration membrane employed in comparative example 1 is different from example 1 in that a cleaning agent fatty alcohol-polyoxyethylene ether solution is not used.
Comparative example 2
The method for preparing a guided tissue regeneration membrane employed in comparative example 2 is different from example 1 in that the cleaning agent used is: 0.25% Triton X-100 and 0.25% sodium deoxycholate.
Comparative example 3
The method for preparing a guided tissue regeneration membrane employed in comparative example 3 is different from example 1 in that the cleaning agent used is: 0.25% Triton X-100 and 0.5% EDTA.
Comparative example 4
The method for preparing a guided tissue regeneration membrane employed in comparative example 4 is different from example 1 in that acetic acid is used at a concentration of 0.5M acetic acid.
Comparative example 5
The method for preparing a guided tissue regeneration membrane employed in comparative example 5 is different from example 1 in that oxalic acid is used at a concentration of 0.5M oxalic acid.
Comparative example 6
The method for preparing a guided tissue regeneration membrane employed in comparative example 6 is different from example 1 in that citric acid is not used.
Comparative example 7
The method for preparing a guided tissue regeneration membrane employed in comparative example 7 is different from example 1 in that a solution of a short peptide is not used.
Comparative example 8
The method for preparing the guided tissue regeneration membrane used in this comparative example is different from example 1 in the lyophilization step and parameters, and the lyophilization step and specific parameters of comparative example 8 are shown in the following table:
table 4: lyophilization step and specific parameters
Comparative example 9
The method for preparing the guided tissue regeneration membrane used in this comparative example is different from example 1 in the lyophilization step and parameters, and the lyophilization step and specific parameters of comparative example 9 are shown in the following table:
table 5: lyophilization step and specific parameters
Comparative example 10
Comparative example 10 differs from example 1 in that a bovine small intestine submucosa film was used in this comparative example.
Comparative example 11
Comparative example 11 differs from example 1 in that bovine peritoneum was used in this comparative example.
Comparative example 12
Comparative example 12 differs from example 1 in that a bovine membrane was used in this comparative example.
Comparative example 13
Comparative example 13 differs from example 1 in the following manner:
(1) The cleaning agent used is: 0.25% triton x-100 and 0.25% sodium deoxycholate;
(2) Citric acid is not used;
(3) Short peptide solutions were not used;
(4) The lyophilization procedure was: the lyophilization procedure in table 5.
Experimental example
1. Decellularization effect detection
Pericardial samples prepared in each example and comparative example were sectioned in paraffin, and compared for residual cells in pericardial tissue by HE staining method, and the results are shown in the following table:
table 6: HE staining results for each sample
As can be seen from the above table, the present invention can effectively remove cells from pericardial tissue by using the decellularized detergent as compared with the comparative example. This is probably due to the fact that the fatty alcohol-polyoxyethylene ether can degrade the phospholipid bilayer of the cell membrane, which is the rupture of the cell membrane, thereby achieving the purpose of removing the cells. Comparative example 1 did not remove cells because no detergent was used, and comparative examples 2 to 3 used a decellularization treatment agent, but the effect was inferior to that of examples in the same time, and it was found that the decellularization detergent of the present invention was more effective in removing cells.
2. Porosity detection
Pericardium films prepared in each example and comparative example were cut into samples of the same size 5cm x 5cm, and their porosities were measured using a 3H-2000TD helium specific gravity analyzer, with the following results:
table 7: results of the porosity detection of each sample
| Group of | Porosity (%) |
| Example 1 | 95.2 |
| Example 2 | 93.4 |
| Example 3 | 92.7 |
| Example 4 | 94.5 |
| Example 5 | 93.1 |
| Comparative example 4 | 61.7 |
| Comparative example 5 | 67.3 |
| Comparative example 6 | 60.4 |
As can be seen from the table above, the use of citric acid in the present invention increases the porosity of pericardial tissue and provides more room for cells to grow, as compared to the comparative example. The comparative examples 4 and 5 used other kinds of acids, and did not significantly improve the porosity, although the porosity was increased, as compared to the comparative example 6, which did not use the acid treatment.
3. Cell adhesion test
Pericardial samples of each example and comparative example were prepared as 5 cm. Times.5 cm-sized samples and placed in a 1X 10 5 And incubating the cells in the 3T3 cell culture solution with good culture state for 2 hours at 37 ℃, and washing the sample with PBS to remove redundant cells. Cells attached to the sample were digested with pancreatin and collected, and absorbance OD was measured at a wavelength of 450nm using LDH kit (Roche, 04744926001) 450nm Higher absorbance values indicate higher cell content. The results are shown in the following table:
table 8: cell adhesion Activity results
| Group of | OD 450nm |
| Example 1 | 1.6 |
| Example 2 | 1.5 |
| Example 3 | 1.4 |
| Example 4 | 1.5 |
| Example 5 | 1.3 |
| Comparative example 7 | 0.6 |
As can be seen from the above table, compared with the comparative example, the invention combines the short peptide with cell adhesion in the pericardial tissue, enhances the cell adhesion of tissue material, is beneficial to the growth of cells along the tissue and promotes the healing of wounds.
4. Mechanical test data comparison
The guided tissue regeneration membranes prepared by the methods of the examples and the comparative examples are subjected to mechanical tests, and the specific test method comprises the following steps: the prepared samples were prepared into rectangular test bars (5 samples were taken for each example or comparative example) of 1cm×5cm, and the maximum tensile strength was measured on a mechanical tester at room temperature and pressure with a sensor 1000N at a tensile strain rate of 1mm/min, with the results shown in the following table:
table 9: guiding tissue regeneration membrane mechanical test data comparison result
As can be seen from table 9, the biomechanical strength of the pericardium prepared by the method in the example is significantly stronger than that of the comparative example, and the comparative examples 8 and 9 use the same pericardium material as the example, but due to the difference of the lyophilization procedure, the arrangement of the collagen structure in the pericardium material is affected, and a loose layer and a dense layer cannot be formed, thereby affecting the biomechanical properties of the guided tissue regeneration membrane. Comparative examples 10 to 12 used other animal membrane tissue materials in the same manner as in example 1, and found that it was not possible to obtain a tissue regeneration membrane having a loose layer and a dense layer, resulting in a decrease in mechanical properties.
5. Animal experiment
And (3) molding: each animal was fixed and the limbal intravenous injection of anesthetic to rabbits given reduced corneal reflex. The rabbit mandibular lower edge area is shaved, the conventional disinfection, under the aseptic condition, the mandibular lower edge of the mandibular angle area is made into skin incision, and the mandibular angle front edge operation area is fully exposed. Circular bone defects 8mm in diameter and 2mm deep were prepared on both sides of the rabbit mandible at the mandibular lower margin using a dental turbine.
The defect areas are treated by the following steps: example 1 pericardium and comparative example 13 pericardium were covered with physiological saline. The soft tissue flap was sufficiently free to close the incision in layers without tension.
After 3 months, the results of the comparison are shown in FIG. 1, and the experimental anatomic diagram shows that the bone defect using the example group membrane has substantially healed (FIG. 1A), while the circular bone defect using the comparative example 13 membrane has significantly healed (FIG. 1B). The results of the software automatic calculation of the volume and the bone density of the new bone of the comparative example and the example show that the guiding tissue regeneration membrane of the invention has remarkable effect in guiding tissue regeneration and can improve the volume and the bone density of the new bone.
Table 10: bone volume and bone density of new bone
6. Histological section comparison
As shown in fig. 2, it can be seen from the three-dimensional reconstruction and the tissue section that the guided tissue regeneration membrane of the present invention has an obvious effect in guiding bone tissue regeneration, and it can be seen that the mechanical properties thereof are changed during the preparation of the pericardium, thereby affecting the end use effect of the pericardium.
In summary, the invention prepares a guided tissue regeneration membrane by taking a pericardium as a tissue material, and improves the preparation method in a certain way. For example: the invention provides a novel decellularized cleaning agent, which uses 0.1-0.8% of fatty alcohol polyoxyethylene ether solution to treat cells on pericardial tissues, and has better effect, shorter decellularized treatment time and less cell residue compared with other reagents. The invention provides more room for cell growth by using citric acid to treat the pericardial tissue material to increase the porosity of the material. The invention also crosslinks short peptide which promotes cell adhesion on the pericardial tissue material, enhances the cell adhesion of the tissue material, and ensures that cells grow along the tissue material to be beneficial to the healing of wounds. According to the invention, a loose layer and a compact layer double-layer structure is obtained through a subsequent specific freeze-drying procedure, and the structure can improve the mechanical property of the regeneration film and accelerate the growth speed and bone density of the new bone in the bone defect area. However, when the production method of the present invention was applied to the production of a regenerated membrane from other animal tissue membranes, it was found that the regenerated membrane having the same structure as that of the example could not be obtained. Therefore, the invention sets a specific freeze-drying procedure aiming at the pericardium, so that the regenerated membrane from the pericardium obtains a specific loose layer and compact layer double-layer structure, thereby improving the mechanical property of the regenerated membrane, accelerating the growth speed of new bone in the bone defect area and accelerating the healing of the defect area.
Examples 6 and 7 were prepared by using pericardium of other animals, and had the same effects as examples 1 to 5, and will not be described here again.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (10)
1. A method for preparing a guided tissue regeneration membrane, comprising the steps of:
(1) Repeatedly freezing and thawing after removing the redundant impurities of the pericardium;
(2) Performing decellularization treatment on the pericardium in the step (1);
(3) Cleaning the pericardium of the step (2) by using a cleaning agent;
(4) Treating the pericardium of step (3) with citric acid;
(5) Immersing the pericardium of step (4) in a solution containing a short peptide;
(6) And (5) freeze-drying the pericardium in the step (5).
2. The method of claim 1, wherein the repeated freeze-thawing specific operation of step (1) is: and (3) placing the pericardium after removing the redundant impurities in PBS liquid, freezing for 2-4 hours at the temperature of-80 ℃ to-50 ℃, then taking out the pericardium and cleaning in a water bath for 30-60min, and repeatedly freezing and thawing for 2-4 times.
3. The method of claim 1, further comprising sterilizing the pericardium of step (1) prior to step (2); the specific operation of the disinfection is as follows: taking out pericardium, immersing in 75% (v/v) alcohol for 2-4 hr, and changing liquid once; immersing the processed pericardium into PBS liquid, placing in a shaking table, and cleaning at a speed of 60-120 rpm for 4-6h, and changing the PBS liquid every 2h.
4. The method of claim 1, wherein the step (2) decellularizing treatment is specifically performed as: putting the treated pericardium into pancreatin solution with mass-volume percentage concentration of 0.2% -0.5% for cell removal treatment for 12-24h, and rinsing with purified water after treatment.
5. The method of claim 1, wherein the step (3) of cleaning treatment is specifically performed as: immersing the pericardium in a cleaning agent and cleaning for 8-16h in a normal temperature shaking table at a speed of 60-120 rpm; the cleaning agent is 0.1% -0.8% (v/v) of fatty alcohol polyoxyethylene ether solution.
6. The preparation method according to claim 1, wherein the citric acid treatment in the step (4) comprises the following specific steps: immersing the pericardium in citric acid solution with concentration of 0.1-0.8M, and oscillating for 0.5-2 h at normal temperature.
7. The method of claim 1, further comprising washing the pericardium of step (4) prior to step (5); the cleaning specific operation is as follows: taking out pericardium, immersing in PBS liquid, and placing in a shaker, and cleaning at 60-120 rpm for 8-12 hr, wherein PBS liquid is replaced every 4 hr.
8. The preparation method according to claim 1, wherein the step (5) is specifically: placing the pericardium in PBS (phosphate buffer solution) containing 0.1-0.8 mg/mL of carbodiimide and 0.1-1 mg/mL of succinimide, washing with PBS after reacting for 10-30 min, placing the pericardium in PBS (phosphate buffer solution) containing short peptide with concentration of 0.1-1 mg/mL, incubating for 2-4h at normal temperature, and rinsing with pure water to remove redundant short peptide; the short peptide is composed of glycine, phenylalanine, hydroxyproline, glutamic acid and arginine.
9. The method of claim 1, wherein the step (6) lyophilization is specifically performed as:
the freeze-drying comprises a pre-freezing stage and a sublimation drying stage;
the pre-freezing stage comprises a first pre-freezing stage and a second pre-freezing stage;
the temperature of the first pre-freezing stage is between-5 ℃ and-2 ℃, the time is between 10 and 60 minutes, and the heat preservation time is between 20 and 60 minutes;
the temperature of the second pre-freezing stage is between 50 ℃ below zero and 20 ℃ below zero, the time is 20 to 60 minutes, and the heat preservation time is 40 to 60 minutes;
the sublimation drying stage comprises a first sublimation drying stage, a second sublimation drying stage and a third sublimation drying stage;
the temperature of the first sublimation drying stage is between-5 ℃ and-2 ℃, the time is 40-120 min, the heat preservation time is 200-400 min, and the vacuum degree is set to be 20-40 Pa;
the temperature of the second sublimation drying stage is 3-8 ℃, the time is 20-120 min, the heat preservation time is 20-120 min, and the vacuum degree is set to be 20-40 Pa;
the temperature of the third sublimation drying stage is 10 ℃ to 20 ℃, the time is 20 to 60min, the heat preservation time is 40 to 120min, and the vacuum degree is set to be 20 to 40Pa.
10. A guided tissue regeneration membrane prepared by the preparation method according to any one of claims 1 to 9, wherein the guided tissue regeneration membrane comprises collagen; it has a multi-layer structure including a porous layer and a dense layer.
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