CN111249534A - Bioactive scaffold capable of promoting synchronous repair and regeneration of wound tissues and preparation method thereof - Google Patents
Bioactive scaffold capable of promoting synchronous repair and regeneration of wound tissues and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a bioactive scaffold for promoting synchronous repair and regeneration of wound tissues, which comprises at least two bioactive scaffolds formed by stacking or splicing three-dimensional space forms; the support materials adopted by each three-dimensional space form are different and at least comprise biomedical polymer hydrogel; after the bioactive scaffold is assembled, the bioactive scaffold is crosslinked and washed by a crosslinking agent; the scaffold is a bioactive scaffold which is constructed by different material proportions and space forms and has the function of promoting wound tissue synchronous repair.
Description
Technical Field
The invention relates to the field of tissue engineering and regenerative medicine, in particular to a bioactive scaffold for promoting synchronous repair and regeneration of wound tissues and a preparation method thereof.
Background
The wound surface injury is a common high-incidence disease in clinic, which not only harms the health of patients, but also seriously affects the life quality of the patients. With the continuous improvement of living standard, people not only hope that the wound surface is quickly covered to reduce the occurrence of complications, but also hope that the wound surface can recover the original appearance and function to achieve the perfect repair and regeneration of tissues. To achieve perfect tissue repair, synchronous regeneration of multiple tissue cells at the damaged part must be achieved. At the present stage, scholars in the basic research field reprogram somatic cells by using various small molecule compounds, transcription factors and the like; the induction of reprogramming pluripotent stem cells (iPS) and the construction of tissue repair biomaterials using tissue engineering 3D printing have made great progress in regenerative medicine. It should also be seen that regenerative medicine has evolved at a distance from the goal of perfect repair of tissue damage and regeneration, and from the patient's requirements.
The bioglass is an artificial biomaterial, can be organically combined with human tissues, and has good biocompatibility. With the continuous and deep research, the nanometer bioglass is widely applied in the fields of dentistry, bone repair, drug carriers and wound repair. But it has the disadvantages of low mechanical strength, large brittleness and the like. Gelatin is a product obtained by partial hydrolysis of collagen, so that the gelatin has incomparable biocompatibility and bioactivity compared with other synthetic materials; and because of its unique physicochemical properties, gelatin is widely used in the field of biomaterials. The existing active scaffold made of a single material of bioglass or gelatin cannot meet the requirements of different cell growth; moreover, most of the existing tissue repair scaffolds are single-layer porous, tissues are easy to grow in, the anti-adhesion effect is long, and the invasion of tissues on different sides inhibits the repair process, so that the treatment effect is reduced.
Disclosure of Invention
The invention aims to provide a bioactive scaffold capable of promoting synchronous repair and regeneration of wound tissues, which is constructed by different material proportions and spatial forms and has the function of promoting synchronous repair of the wound tissues.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a biological active scaffold for promoting synchronous repair and regeneration of wound tissue comprises at least two biological active scaffolds formed by stacking or splicing three-dimensional space forms; the support materials adopted by each three-dimensional space form are different and at least comprise biomedical polymer hydrogel; the bioactive scaffolds were cross-linked by a cross-linking agent and washed after assembly.
In some improvements, the bioactive scaffold is in a double-layer three-dimensional form or a multi-layer three-dimensional form formed by stacking up and down.
In one improvement, the bioactive scaffold is in the form of a double-layer cylinder stacked up and down, wherein the upper layer cylinder is made of 15-25 wt% gelatin solution, and the lower layer cylinder is made of 15-25 wt% gelatin solution and 3-10 wt% silicate bioglass by mixing.
In another improvement scheme, the bioactive scaffold is in a three-layer column body form stacked up and down, wherein the upper-layer column body is prepared by mixing 20-30 wt% of chitosan solution and 15-25 wt% of sodium alginate solution, and the aperture ratio is 60-90%; the middle-layer cylinder is made of 20-30 wt% of chitosan solution, and the aperture ratio is 0-20%; the lower column body adopts 20-30 wt% of chitosan solution, 15-25 wt% of sodium alginate solution and 3-10 wt% of silicate bioglass, and the aperture ratio is 60-90%.
The invention also discloses a preparation method of the bioactive scaffold for promoting synchronous repair and regeneration of wound tissues, which comprises the following steps:
1) designing the space form and material ratio of the bracket according to different wound tissue types;
2) preparing solutions in all three-dimensional space forms by using the selected material ratio, and preparing all three-dimensional space forms in a gel state in a low-temperature environment;
3) assembling all three-dimensional space forms in a gel state into an integral structure to obtain an initial bioactive scaffold;
4) pouring a cross-linking agent solution on the obtained initial bioactive scaffold, cross-linking the initial bioactive scaffold, and washing;
5) and (3) freeze-drying the washed bioactive scaffold by using a freeze dryer, and removing water to obtain the bioactive scaffold.
The bioactive scaffold for promoting synchronous repair and regeneration of wound tissues and the preparation method thereof provided by the invention have the following advantages:
the adopted biological scaffold has diversity of space forms and material proportions, and different material proportions and space forms can be selected according to different tissue types and forms of the wound surface, so that synchronous repair and regeneration of the wound surface tissue are better promoted.
The invention also provides a bioactive bracket with an interlayer structure, the brackets on two sides of the interlayer promote different tissues to be repaired, the interlayer connection is tight, the interlayer structure is used as a barrier to avoid the mutual invasion of the tissues on two sides, the three-dimensional culture space required by the growth of tissue cells can be better simulated, and the function maintenance of different tissues is more facilitated.
The invention provides a method for constructing a bioactive scaffold by utilizing different material ratios and different space forms, which corresponds to the requirements of different tissues for repair and regeneration, thereby better promoting the synchronous repair and regeneration of wound tissues and providing a new visual angle for regenerative medicine and tissue engineering.
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In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without inventive efforts.
FIG. 1 is a two-layer three-dimensional bioactive scaffold according to an embodiment of the present invention;
FIG. 2 is a fluorescent image of experimental example of two-layer bioactive scaffold inoculated with mouse fibroblasts;
FIG. 3 is a fluorescence image of mouse fibroblasts seeded with three layers of bioactive scaffolds in an experimental example.
Detailed Description
In order to make the purpose, technical solution and beneficial effects of the present application more clear and more obvious, the present application is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.
A biological active scaffold for promoting synchronous repair and regeneration of wound tissue comprises at least two biological active scaffolds formed by stacking or splicing three-dimensional space forms; the support materials adopted by each three-dimensional space form are different and at least comprise biomedical polymer hydrogel; the bioactive scaffolds were cross-linked by a cross-linking agent and washed after assembly.
The biomedical polymer hydrogel comprises one or more of gelatin, sodium alginate, chitosan, agarose, gellan gum, silk fibroin, collagen or hyaluronic acid. At least one of the scaffold materials of the three-dimensional space of the bioactive scaffold also comprises one or the combination of more than two of borosilicate bioglass, silicate bioglass, calcium phosphate bioglass, bioactive glass, bioceramic, active trace elements and polyacrylamide.
The bioactive bracket is in a double-layer three-dimensional space form and a multi-layer three-dimensional space form formed by adopting an up-and-down stacking mode. Or, the three-dimensional light source is formed by splicing different parts around the same axis, such as a five-pointed star solid structure in a scattering state or the like. It should be noted that the bioactive scaffold is provided with different morphological structures according to different wound positions to better match with the part to be repaired.
The preparation method of the bioactive scaffold comprises the following steps:
1) designing the space form and material ratio of the bracket according to different wound tissue types;
2) preparing solutions in all three-dimensional space forms by using the selected material ratio, and preparing all three-dimensional space forms in a gel state in a low-temperature environment;
3) assembling all three-dimensional space forms in a gel state into an integral structure to obtain an initial bioactive scaffold;
4) pouring a cross-linking agent solution on the obtained initial bioactive scaffold, cross-linking the initial bioactive scaffold, and washing;
5) and (3) freeze-drying the washed bioactive scaffold by using a freeze dryer, and removing water to obtain the bioactive scaffold.
The following is a detailed description of embodiments according to the present invention.
Example 1
The bioactive scaffold is in a double-layer cylinder shape stacked up and down, as shown in figure 1;
the upper layer cylinder is made of 15 wt% gelatin solution;
the lower cylinder is prepared by mixing 15 wt% of gelatin solution and 3 wt% of silicate bioglass;
the preparation method comprises the following steps:
10) preparing upper-layer cylindrical liquid: mixing gelatin and deionized water in a weight ratio of 1:15-25, shaking for two hours at 37 ℃ to ensure that the gelatin is completely fused and uniformly dispersed, and then standing for two hours at room temperature to completely discharge bubbles; then carrying out pasteurization to obtain a gelatin solution;
11) preparing lower-layer cylindrical liquid: mixing gelatin with deionized water in a weight ratio of 1:15-25 to prepare a gelatin solution; mixing silicate bioglass with deionized water in a weight ratio of 1:3-10 to prepare a silicate bioglass solution; mixing the gelatin solution and the silicate bioglass solution at 37 ℃ and shaking for two hours to ensure that the gelatin solution and the silicate bioglass solution are completely fused and uniformly dispersed, and then standing for two hours at room temperature to completely discharge bubbles; then, sterilizing the solution by pasteurization to obtain a mixed solution of gelatin and silicate bioglass;
12) extracting the gelatin solution obtained in the step 10) by using a medical injector, and placing the gelatin solution in an environment of 4 ℃ for 10 minutes to obtain a gelatin cylinder in a gel state;
13) extracting the mixed solution obtained in the step 11) again by using a medical injector, placing the mixed solution in an environment of 4 ℃ for 10 minutes to enable the components of the mixed solution to be in a gel state, then vertically stacking the components with the end face of a gelatin cylinder in the gel state, and completely bonding the components with the end face of the gelatin cylinder to obtain an integrated bioactive bracket;
14) and (3) taking a 1% glutaraldehyde solution to the integrated bioactive scaffold obtained in the step 13), crosslinking, repeatedly soaking and cleaning the bioactive scaffold by using a 1% glutamic acid solution after crosslinking, and removing residual glutaraldehyde to obtain the bioactive scaffold.
Example 2
The bioactive bracket is in a double-layer cylinder shape stacked up and down;
the upper cylinder is made of 30 wt% gelatin solution;
the lower cylinder is prepared by mixing 30 wt% of gelatin solution and 10 wt% of silicate bioglass;
the preparation is as described in example 1.
Example 3
The bioactive bracket is in a double-layer cylinder shape stacked up and down;
the upper layer cylinder is made of a gelatin solution with the weight of 20 percent;
the lower cylinder is prepared by mixing 20 wt% of gelatin solution and 5 wt% of silicate bioglass;
the preparation method comprises the following steps:
10) preparing upper-layer cylindrical liquid: mixing gelatin and deionized water in a weight ratio of 1:15-25, shaking for two hours at 37 ℃ to ensure that the gelatin is completely fused and uniformly dispersed, and then standing for two hours at room temperature to completely discharge bubbles; then carrying out pasteurization to obtain a gelatin solution;
11) preparing lower-layer cylindrical liquid: mixing gelatin with deionized water in a weight ratio of 1:15-25 to prepare a gelatin solution; mixing silicate bioglass with deionized water in a weight ratio of 1:3-10 to prepare a silicate bioglass solution; mixing the gelatin solution and the silicate bioglass solution at 37 ℃ and shaking for two hours to ensure that the gelatin solution and the silicate bioglass solution are completely fused and uniformly dispersed, and then standing for two hours at room temperature to completely discharge bubbles; then, sterilizing the solution by pasteurization to obtain a mixed solution of gelatin and silicate bioglass;
12) extracting the gelatin solution obtained in the step 10) by using a medical injector, and placing the gelatin solution in an environment of 4 ℃ for 10 minutes to obtain a gelatin cylinder in a gel state;
13) extracting the mixed solution obtained in the step 11) again by using a medical injector, placing the mixed solution in an environment of 4 ℃ for 10 minutes to enable the components of the mixed solution to be in a gel state, then vertically stacking the components with the end face of a gelatin cylinder in the gel state, and completely bonding the components with the end face of the gelatin cylinder to obtain an integrated bioactive bracket;
14) taking 1% glutaraldehyde solution to the integrated bioactive scaffold obtained in the step 13), crosslinking, repeatedly soaking and cleaning the bioactive scaffold with 1% glutamic acid solution after crosslinking, and removing residual glutaraldehyde to obtain the bioactive scaffold;
15) and freeze-drying the bioactive scaffold in a low-temperature freeze dryer for 3 days, and removing redundant water in the scaffold to complete the construction of the porosity of the bioactive scaffold.
Example 4
The bioactive bracket is in a three-layer cylinder shape stacked up and down;
the upper layer column body is prepared by mixing 20 wt% of chitosan solution and 15 wt% of sodium alginate solution;
the middle-layer cylinder is made of 20 wt% of chitosan solution;
the lower column adopts 20 wt% of chitosan solution, 15 wt% of sodium alginate solution and 3 wt% of silicate bioglass.
The preparation method comprises the following steps:
20) preparing lower column liquid: mixing chitosan with 1% (v/v) acetic acid solution, and slowly stirring at 37 deg.C for two hours to completely fuse and uniformly disperse chitosan to obtain 20-30% wt chitosan solution; mixing sodium alginate with 25mg/mL NaCl solution, and slowly stirring at 37 deg.C for two hours to completely fuse and uniformly disperse sodium alginate to obtain 15-25% wt sodium alginate solution; mixing silicate bioglass with deionized water in a weight ratio of 1:3-10 to prepare a silicate bioglass solution; mixing chitosan solution, sodium alginate solution and silicate bioglass solution at 37 deg.C, shaking for two hours to make the solution completely fuse and uniformly disperse, slowly stirring for 6 hours, then pasteurizing, adding sodium dodecyl sulfate, stirring for 5-10min to obtain upper layer column solution; wherein, the amount of the sodium dodecyl sulfate added into each 10mL of the mixed solution is 10 mg;
21) preparing middle-layer column liquid: mixing chitosan with 1% (v/v) acetic acid solution, slowly stirring at 37 deg.C for two hours to completely fuse and uniformly disperse chitosan, standing at room temperature for two hours to completely discharge bubbles, and pasteurizing to obtain middle column solution;
22) preparing upper layer column liquid: mixing chitosan with 1% (v/v) acetic acid solution, and slowly stirring at 37 deg.C for two hours to completely fuse and uniformly disperse chitosan to obtain 20-30% wt chitosan solution; mixing sodium alginate with 25mg/mL NaCl solution, and slowly stirring at 37 deg.C for two hours to completely fuse and uniformly disperse sodium alginate to obtain 15-25% wt sodium alginate solution; mixing 1:1 amount of chitosan solution and sodium alginate solution, slowly stirring for 6 hr, pasteurizing, adding sodium dodecyl sulfate, and stirring for 5-10min to obtain upper column solution; wherein, the amount of the sodium dodecyl sulfate added into each 10mL of the mixed solution is 10 mg;
23) extracting the solution of the lower column body in the step 20) by using a medical injector, and placing the solution in an environment of 4 ℃ for 10 minutes to obtain a lower column body in a gel state;
24) extracting the solution of the middle-layer cylinder in the step 21) again by using a medical injector, placing the solution in an environment at 4 ℃ for 10 minutes to enable the components of the mixed solution to be in a gel state, then stacking the mixed solution to the upper end face of the lower-layer cylinder in the gel state, and completely bonding the two to obtain a double-layer bioactive scaffold;
25) extracting the upper layer column solution obtained in the step 22) again by using a medical injector, placing the upper layer column solution in an environment at 4 ℃ for 10 minutes to enable the components of the mixed solution to be in a gel state, then stacking the mixed solution to the upper end face of the middle layer column in the gel state, and completely bonding the two to obtain a three-layer bioactive scaffold;
26) and (3) taking a 1% glutaraldehyde solution to the integrated bioactive scaffold obtained in the step 25), crosslinking, repeatedly soaking and cleaning the bioactive scaffold by using a 1% glutamic acid solution after crosslinking, and removing residual glutaraldehyde to obtain the bioactive scaffold.
The aperture ratio of the upper layer column of the prepared bioactive scaffold is 70.6 percent, and the aperture size is 890-2600 mu m; the aperture ratio of the middle-layer cylinder is 2.51 percent, and the aperture size is 20-300 mu m; the lower column has a pore diameter of 67.4% and a pore size of 1200 and 2700 μm.
Example 5
The bioactive bracket is in a three-layer cylinder shape stacked up and down;
the upper layer column body is prepared by mixing 30 wt% of chitosan solution and 25 wt% of sodium alginate solution;
the middle-layer column body is made of 30 wt% of chitosan solution;
the lower column adopts 30 wt% of chitosan solution, 25 wt% of sodium alginate solution and 10 wt% of silicate bioglass.
The preparation method comprises the following steps: as shown in example 4, the difference is that: 20) when preparing the lower-layer column solution, 15mg of sodium dodecyl sulfate is added into every 10mL of mixed solution; 22) when the lower column solution was prepared, 15mg of sodium dodecylsulfate was added per 10mL of the mixture.
The aperture ratio of the upper layer column of the prepared bioactive scaffold is 78.1 percent, and the aperture size is 850-; the aperture ratio of the middle-layer cylinder is 1.62 percent, and the aperture size is 10-280 mu m; the lower column has a pore size of 860 and 3000 μm, and a porosity of 76.6%.
Example 6
The bioactive bracket is in a three-layer cylinder shape stacked up and down;
the upper layer column body is prepared by mixing 25 wt% of chitosan solution and 20 wt% of sodium alginate solution;
the middle-layer column body is made of 25 wt% of chitosan solution;
the lower column adopts 25 wt% of chitosan solution, 20 wt% of sodium alginate solution and 5 wt% of silicate bioglass.
The preparation method comprises the following steps: as shown in example 4, the difference is that: 20) when preparing the lower column solution, 13.2mg of sodium dodecyl sulfate is added into each 10mL of mixed solution; 22) when the lower column solution was prepared, 13.0mg of sodium dodecylsulfate was added per 10mL of the mixture.
The aperture ratio of the upper layer column of the prepared bioactive scaffold is 81.2 percent, and the aperture size is 900-; the aperture ratio of the middle-layer cylinder is 1.02 percent, and the aperture size is 32-240 mu m; the aperture ratio of the lower column is 88.6%, and the aperture size is 1000-3000 μm.
Test examples the bioactive scaffold of the present invention was inoculated into mouse fibroblasts
1. Bioactive scaffolds employing the double-layer pillar configuration described in example 2
The experimental process comprises the following steps:
1) the integrated bioactive scaffold described in example 2 was constructed.
2) And extracting 24-hour C57 mouse fibroblasts by adopting a differential adherence method, taking P2 exponential phase cells, and preparing a fibroblast solution with the concentration of 1 × 105/ML by using a DMEM high-glucose complete culture medium.
3) Dripping fibroblast solution on the surface of the bioactive bracket
4) Placing the biological active scaffold inoculated with the cells in a DMEM high-glucose complete culture medium for culture, and changing the culture solution every 3 days.
5) The state and shape of the fibroblasts were observed under a fluorescence microscope at 3-day and 7-day time points.
The experimental results are as follows: FIG. 2 shows the fluorescence results at the 7 day time point, showing that mouse fibroblasts stretch well within the bioactive scaffold.
2. Bioactive scaffolds employing the three-layer pillar configuration described in example 5
The experimental process comprises the following steps:
1) the integrated bioactive scaffold described in example 5 was constructed.
2) And extracting 24-hour C57 mouse fibroblasts by adopting a differential adherence method, taking P2 exponential phase cells, and preparing a fibroblast solution with the concentration of 1 × 105/ML by using a DMEM high-glucose complete culture medium.
3) Dripping fibroblast solution on the upper and lower surfaces of the three-layer bioactive scaffold
4) Placing the biological active scaffold inoculated with the cells in a DMEM high-glucose complete culture medium for culture, and changing the culture solution every 3 days.
5) The state and shape of the fibroblasts were observed under a fluorescence microscope at 3-day and 7-day time points.
The experimental results are as follows: FIG. 3 is a partial view of the upper and middle layer junctions, showing fluorescence results at 7 day time points, showing that mouse fibroblasts stretch well within the bioactive scaffold, while the middle sandwich porosity is low and cell invasion is low.
The above-described embodiments do not limit the scope of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the above-described embodiments should be included in the protection scope of the technical solution.
Claims (10)
1. A bioactive scaffold for promoting synchronous repair and regeneration of wound tissues is characterized by comprising at least two bioactive scaffolds formed by stacking or splicing three-dimensional space forms; the support materials adopted by each three-dimensional space form are different and at least comprise biomedical polymer hydrogel; the bioactive scaffolds were cross-linked by a cross-linking agent and washed after assembly.
2. The bioactive scaffold for promoting synchronous repair and regeneration of wound tissues as claimed in claim 1, wherein said biomedical polymer hydrogel comprises one or more of gelatin, sodium alginate, chitosan, agarose, gellan gum, silk fibroin, collagen or hyaluronic acid;
the scaffold material of the three-dimensional space of at least one bioactive scaffold also comprises one or the combination of more than two of borosilicate bioglass, silicate bioglass, calcium phosphate bioglass, bioactive glass, bioceramic, active trace elements and polyacrylamide.
3. A bioactive scaffold as claimed in claim 1 for use in promoting the synchronous repair and regeneration of wound tissue, wherein the bioactive scaffold is in the form of a double layer three-dimensional space or a multiple layer three-dimensional space formed by stacking up and down.
4. The bioactive scaffold for promoting the synchronous repair and regeneration of wound tissues as claimed in claim 1, wherein said bioactive scaffold is in the form of a double-layered cylinder stacked up and down, wherein the upper layer cylinder is made of 15-25% wt gelatin solution, and the lower layer cylinder is made of 15-25% wt gelatin solution mixed with 3-10% wt silicate bioglass.
5. A bioactive scaffold as claimed in claim 4 which is used to promote the synchronous repair and regeneration of wound tissue, wherein the upper cylinder is made of 20% wt gelatin solution and the lower cylinder is made of 20% wt gelatin solution mixed with 5% wt silicate bioglass.
6. The bioactive scaffold for promoting the synchronous repair and regeneration of wound tissues as claimed in claim 1, wherein the bioactive scaffold is in the form of a three-layer column stacked up and down, wherein the upper layer column is prepared by mixing 20-30% wt of chitosan solution and 15-25% wt of sodium alginate solution, and the aperture ratio is 60-90%; the middle-layer cylinder is made of 20-30 wt% of chitosan solution, and the aperture ratio is 0-20%; the lower column body adopts 20-30 wt% of chitosan solution, 15-25 wt% of sodium alginate solution and 3-10 wt% of silicate bioglass, and the aperture ratio is 60-90%.
7. A preparation method of a bioactive scaffold for promoting synchronous repair and regeneration of wound tissues is characterized by comprising the following steps:
1) designing the space form and material ratio of the bracket according to different wound tissue types;
2) preparing solutions in all three-dimensional space forms by using the selected material ratio, and preparing all three-dimensional space forms in a gel state in a low-temperature environment;
3) assembling all three-dimensional space forms in a gel state into an integral structure to obtain an initial bioactive scaffold;
4) pouring a cross-linking agent solution on the obtained initial bioactive scaffold, cross-linking the initial bioactive scaffold, and washing;
5) and (3) freeze-drying the washed bioactive scaffold by using a freeze dryer, and removing water to obtain the bioactive scaffold.
8. A method for preparing a bioactive scaffold for promoting synchronous repair and regeneration of wound tissues, which is used for preparing the bioactive scaffold with the double-layer cylinder shape of claim 4, and comprises the following steps:
10) preparing upper-layer cylindrical liquid: mixing gelatin and deionized water in a weight ratio of 1:15-25, shaking for two hours at 37 ℃ to ensure that the gelatin is completely fused and uniformly dispersed, and then standing for two hours at room temperature to completely discharge bubbles; then carrying out pasteurization to obtain a gelatin solution;
11) preparing lower-layer cylindrical liquid: mixing gelatin with deionized water in a weight ratio of 1:15-25 to prepare a gelatin solution; mixing silicate bioglass with deionized water in a weight ratio of 1:3-10 to prepare a silicate bioglass solution; mixing the gelatin solution and the silicate bioglass solution at 37 ℃ and shaking for two hours to ensure that the gelatin solution and the silicate bioglass solution are completely fused and uniformly dispersed, and then standing for two hours at room temperature to completely discharge bubbles; then, sterilizing the solution by pasteurization to obtain a mixed solution of gelatin and silicate bioglass;
12) extracting the gelatin solution obtained in the step 10) by using a medical injector, and placing the gelatin solution in an environment of 4 ℃ for 10 minutes to obtain a gelatin cylinder in a gel state;
13) extracting the mixed solution obtained in the step 11) again by using a medical injector, placing the mixed solution in an environment of 4 ℃ for 10 minutes to enable the components of the mixed solution to be in a gel state, then vertically stacking the components with the end face of a gelatin cylinder in the gel state, and completely bonding the components with the end face of the gelatin cylinder to obtain an integrated bioactive bracket;
14) and (3) taking a 1% glutaraldehyde solution to the integrated bioactive scaffold obtained in the step 13), crosslinking, repeatedly soaking and cleaning the bioactive scaffold by using a 1% glutamic acid solution after crosslinking, and removing residual glutaraldehyde to obtain the bioactive scaffold.
9. The method for preparing a bioactive scaffold for promoting synchronous repair and regeneration of wound tissue as claimed in claim 8, wherein the step 14) is followed by the following steps:
15) and freeze-drying the bioactive scaffold in a low-temperature freeze dryer for 3 days, and removing redundant water in the scaffold to complete the construction of the porosity of the bioactive scaffold.
10. A method for preparing a bioactive scaffold capable of promoting synchronous repair and regeneration of wound tissues, which is used for preparing the bioactive scaffold in the form of a three-layer cylinder of claim 6, and comprises the following steps:
20) preparing lower column liquid: mixing chitosan with 1% (v/v) acetic acid solution, and slowly stirring at 37 deg.C for two hours to completely fuse and uniformly disperse chitosan to obtain 20-30% wt chitosan solution; mixing sodium alginate with 25mg/mL NaCl solution, and slowly stirring at 37 deg.C for two hours to completely fuse and uniformly disperse sodium alginate to obtain 15-25% wt sodium alginate solution; mixing silicate bioglass with deionized water in a weight ratio of 1:3-10 to prepare a silicate bioglass solution; mixing chitosan solution, sodium alginate solution and silicate bioglass solution at 37 deg.C, shaking for two hours to make the solution completely fuse and uniformly disperse, slowly stirring for 6 hours, then pasteurizing, adding sodium dodecyl sulfate, stirring for 5-10min to obtain upper layer column solution; wherein, the amount of the sodium dodecyl sulfate added into each 10mL of the mixed solution is 10-15 mg;
21) preparing middle-layer column liquid: mixing chitosan with 1% (v/v) acetic acid solution, slowly stirring at 37 deg.C for two hours to completely fuse and uniformly disperse chitosan, standing at room temperature for two hours to completely discharge bubbles, and pasteurizing to obtain middle column solution;
22) preparing upper layer column liquid: mixing chitosan with 1% (v/v) acetic acid solution, and slowly stirring at 37 deg.C for two hours to completely fuse and uniformly disperse chitosan to obtain 20-30% wt chitosan solution; mixing sodium alginate with 25mg/mL NaCl solution, and slowly stirring at 37 deg.C for two hours to completely fuse and uniformly disperse sodium alginate to obtain 15-25% wt sodium alginate solution; mixing 1:1 amount of chitosan solution and sodium alginate solution, slowly stirring for 6 hr, pasteurizing, adding sodium dodecyl sulfate, and stirring for 5-10min to obtain upper column solution; wherein, the amount of the sodium dodecyl sulfate added into each 10mL of the mixed solution is 10-15 mg;
23) extracting the solution of the lower column body in the step 20) by using a medical injector, and placing the solution in an environment of 4 ℃ for 10 minutes to obtain a lower column body in a gel state;
24) extracting the solution of the middle-layer cylinder in the step 21) again by using a medical injector, placing the solution in an environment at 4 ℃ for 10 minutes to enable the components of the mixed solution to be in a gel state, then stacking the mixed solution to the upper end face of the lower-layer cylinder in the gel state, and completely bonding the two to obtain a double-layer bioactive scaffold;
25) extracting the upper layer column solution obtained in the step 22) again by using a medical injector, placing the upper layer column solution in an environment at 4 ℃ for 10 minutes to enable the components of the mixed solution to be in a gel state, then stacking the mixed solution to the upper end face of the middle layer column in the gel state, and completely bonding the two to obtain a three-layer bioactive scaffold;
26) and (3) taking a 1% glutaraldehyde solution to the integrated bioactive scaffold obtained in the step 25), crosslinking, repeatedly soaking and cleaning the bioactive scaffold by using a 1% glutamic acid solution after crosslinking, and removing residual glutaraldehyde to obtain the bioactive scaffold.
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