CN115845115A - Silk protein-based double-layer material and preparation method and application thereof - Google Patents
Silk protein-based double-layer material and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a silk protein based double-layer material and a preparation method and application thereof. The silk fibroin-based double-layer material is composed of a sponge layer and a film layer. The fibroin-based double-layer material disclosed by the invention has good mechanical property, adhesion property and degradability without toxic and side effects, and can be used as a medical material to be prepared into a medical adhesive patch and the like.
Description
Technical Field
The invention belongs to the fields of natural polymer materials, tissue engineering materials and medical instruments, and particularly relates to a fibroin-based double-layer material as well as a preparation method and application thereof.
Background
Tissue trauma refers to the destruction of human tissue or organs by mechanical forces. In order to repair wound tissues, the implementation of wound closure and reduction fixation is an essential first step in clinical surgery, and is crucial to rapid hemostasis, infection avoidance, secondary injury reduction, tissue remodeling and physiological function recovery. With the rapid development of modern science and medical technology, the adhesive is used for closing wounds by medical tissue engineering products to replace the traditional invasive suture technology, and the adhesive becomes a new trend of clinical medicine.
An ideal medical adhesive not only needs to be safe and nontoxic, but also has the mechanical property matched with the damaged tissue, has good adhesive effect and realizes reliable wound traction and fixed closure. The medical adhesives commonly used in clinic at present are divided into two main types of chemical synthetic adhesives and biological adhesives, including amino acrylate, polyurethane, fibrin glue, chitosan adhesive and the like. The advantage of chemically synthesized adhesives is high adhesive strength, but poor biodegradability and biocompatibility. In recent years, adhesive materials based on biomacromolecules such as protein and polysaccharide attract extensive attention of researchers, but the existing protein-based biomedical adhesive has insufficient mechanical property and weak adhesive strength, and cannot meet the adhesive requirements of different damaged tissues. Therefore, the development of protein-based biomaterials with both mechanical properties and ideal adhesion remains an important direction for the development of surgical medicine, tissue repair engineering and biomaterials.
Silk protein is a protein-based biopolymer extracted from natural silkworm cocoons, has excellent mechanical properties, can be degraded into a non-toxic product by a human body, and has excellent biocompatibility and biosafety. Especially, various processing technologies developed aiming at silk protein greatly broadens the application field of silk protein-based materials, and various novel materials such as silk protein-based scaffolds, films, sponges, microspheres and the like are widely applied in various biomedical fields such as epidermal wound healing, bone tissue regeneration, tympanic membrane repair and the like. Therefore, the silk protein is expected to be an ideal natural raw material for preparing biomedical adhesive materials.
Although there are some reports related to designing silk protein based medical adhesives based on chemical modification, until now, there is still a lack of an efficient and easy-to-implement processing and preparation method and practical commercial products. Therefore, the development of a silk protein medical adhesive material which is portable in production process, can be produced in large quantities and enters practical clinical application is still a practical problem which is not fully solved at present.
Disclosure of Invention
One of the objects of the present invention is to provide a silk protein-based bilayer material.
The invention also aims to provide a preparation method of the silk protein-based bilayer material.
Still another object of the present invention is to provide the use of the silk protein-based bilayer material in the preparation of a medical material.
In one aspect, the present invention provides a method for preparing a silk protein-based bilayer material, the method comprising the steps of:
(1) Respectively preparing silk protein, plasticizer and inorganic salt aqueous solution;
(2) Uniformly mixing the silk protein aqueous solution, the plasticizer aqueous solution and the inorganic salt aqueous solution obtained in the step (1) to obtain a uniform precursor solution 1 of silk protein, plasticizer and inorganic salt;
(3) Preparing the precursor solution 1 into a film form and drying to obtain a film layer;
(4) Uniformly mixing the silk protein aqueous solution, the plasticizer aqueous solution and the inorganic salt aqueous solution obtained in the step (1) to obtain a uniform precursor solution 2 of silk protein, plasticizer and inorganic salt;
(5) And (3) precooling the film layer in the step (3) at the temperature of minus 20 to minus 80 ℃, pouring the precursor solution 2 prepared in the step (4) on the frozen film layer, and freeze-drying to prepare a sponge layer, thereby obtaining the silk protein-based double-layer material with the film and sponge structures.
In some embodiments, the silk protein aqueous solution in step (1) may be prepared according to the following method: adding the crushed silkworm cocoons into a boiling sodium carbonate solution, keeping the solution degummed, rinsing the degummed silk and drying. And then adding the dried degummed silk into a lithium bromide aqueous solution for dissolving. And (4) after the silk fiber is completely dissolved, dialyzing and purifying the solution to obtain silk protein water solution, and storing the silk protein water solution in a refrigerator. The concentration of the silk fibroin can be measured by taking a certain amount of silk fibroin aqueous solution and drying. Preferably, the concentration of the silk fibroin aqueous solution is 45-55mg/mL.
In some embodiments, in the preparation of the silk protein aqueous solution of step (1), the concentration of the sodium carbonate solution is 0.01 to 0.05mol/L, and the degumming treatment time is 5 to 180 minutes.
In some embodiments, in the preparation of the silk fibroin aqueous solution of step (1), the concentration of the lithium bromide aqueous solution is 9.3mol/L, and the dissolution is carried out at 60 ℃ for 4 hours.
In some embodiments, in step (1), the plasticizer is a common class applied to silk proteins in the art, preferably the plasticizer is selected from glycerol, sorbitol, polyethylene glycol. Deionized water can be used for preparing the plasticizer water solution, and the concentration of the plasticizer water solution is 50-1000mg/mL.
In some embodiments, in step (1), the inorganic salt may be selected from calcium chloride, lithium bromide. Deionized water can be adopted to prepare an aqueous solution of inorganic salt, and the concentration of the aqueous solution is 0.01-10mol/L.
In some embodiments, in step (3), the precursor solution 1 prepared in step (2) is transferred to a prefabricated mold to be made into a film form, and dried at a temperature of 10 to 80 ℃ for 12 to 48 hours. Drying may be carried out under atmospheric pressure or under vacuum.
In some embodiments, in step (3), the thin film layer may have a thickness ranging from 30 micrometers to 100 micrometers. For example, the thickness of the thin film layer may be controlled by changing the amount of the precursor solution 1 added to the pre-formed mold, but the present invention is not limited thereto.
In some embodiments, the ratio of each substance in both the film layer and the sponge layer may be the same or different. For example, in the film layer, the mass fraction of the plasticizer is 10 to 50% and the mass fraction of the inorganic salt is 2 to 20%; in the sponge layer, the mass fraction of the dry matter of the plasticizer is 10-50%, and the mass fraction of the dry matter of the inorganic salt is 2-20%.
In some embodiments, in the step (2) and the step (4), when the precursor solution is prepared, the layered structure having different adhesive properties and mechanical properties in the patch can be prepared by controlling the mass ratio of the silk protein, the plasticizer and the salt. For example, when the dry mass fraction of plasticizer in the precursor solution is greater than 20%, the prepared patch layer has excellent toughness and stretchability. When the mass fraction of the dry matter of the inorganic salt in the precursor solution is more than 10%, the prepared patch layer has excellent water solubility and adhesiveness. Further, the precursor solutions 1 and 2 may be the same or different solutions. In the case where precursor solutions 1 and 2 are the same solution (i.e., solutions of the same composition), step (2) and step (4) may be combined into one step.
In some embodiments, in step (5), the temperature of the freeze-drying is between-20 ℃ and-80 ℃. In the case of freeze-drying, the film layer on which the precursor solution 2 is cast may be frozen (for example, at least 12 hours) in a refrigerator (-20 to-80 ℃) and then dried (for example, 24 hours) in a freeze-dryer.
In some embodiments, in step (5), the sponge layer thickness may range from 50 microns to 150 microns. For example, the thickness of the thin film layer may be controlled by varying the amount of the precursor solution 2 added to the pre-formed mold, but the present invention is not limited thereto. In some embodiments, the silk protein-based bilayer material can be in the form of a patch, such as a tissue engineering patch.
In another aspect, the present invention provides a silk protein-based bilayer material prepared by the above method.
In still another aspect, the present invention provides a use of the above-described silk protein-based bilayer material in the preparation of a medical material.
In particular embodiments, the medical material can be a medical adhesive patch, more particularly a medical adhesive bandage.
Advantageous effects
The invention prepares the silk protein based double-layer material by controlling the process conditions and changing the mixing proportion of the raw materials, and the silk protein based double-layer material has good mechanical property, adhesive property and degradability without toxic and side effects.
In conclusion, the present application has the following excellent effects:
(1) The silk protein solution used in the preparation method takes natural silkworm cocoons as raw materials, and has wide sources and proper cost; the added reagents, plasticizer and inorganic salt solution are common chemical industry raw materials and are low in price. The preparation method is simple and easy to operate, the processing process is green and environment-friendly, a preparation process is provided for the silk protein-based medical adhesive material and the development of the silk protein-based medical adhesive material in the practical application of the silk protein-based medical adhesive material in the field of clinical medicine, and the preparation method has feasibility of large-scale production and wide industrialization prospect.
(2) The preparation method provided by the invention is based on regulation and control process conditions, double-layer materials with different macroscopic and microscopic structures are designed for the tissue engineering patch, and the crystallization behavior and degree of each layer of fibroin are influenced by the plasticizer and the inorganic salt ions, so that the mechanical property and water solubility of each layer of the patch are regulated and controlled. The sponge layer can be a water-soluble adhesive layer by regulating the mass ratio of fibroin, plasticizer and salt, is rich in viscosity without chemical reaction, has good biocompatibility, and can be naturally degraded into nontoxic products by a human body.
Drawings
Fig. 1 shows front (left) and back (right) photographs of the silk protein-based bilayer tissue engineering patch prepared in example 1.
Fig. 2 shows a cross-sectional scanning electron micrograph of the fibroin-based bilayer tissue engineering patch prepared in example 1.
FIG. 3 shows an infrared absorption spectrum of the thin film layer of the silk protein-based double-layer tissue engineering patch prepared in example 1.
Fig. 4 shows a tensile property characterization diagram of the thin film layer of the silk protein-based double-layer tissue engineering patch prepared in example 1.
Fig. 5 shows a graph representing the adhesiveness of the silk protein-based bilayer tissue engineering patch prepared in example 1.
Fig. 6 shows a water solubility characterization diagram of the silk protein-based bilayer tissue engineering patch prepared in example 1.
Fig. 7 shows a cytocompatibility characterization diagram of the silk protein-based bilayer tissue engineering patch prepared in example 1.
Detailed Description
The invention is described in further detail below with reference to specific examples, which are provided for illustration only and are not intended to limit the invention in any way.
In this application, references to numbers "1", "2", etc. are used only to distinguish one element or component from another element or component, and have no other meaning.
Example 1
A silk protein-based bilayer patch was prepared according to the following method:
(1) Preparing a film layer: preparing a silk protein solution of 50mg/mL, a glycerol solution of 100mg/mL and a calcium chloride solution of 1mol/L, adding 1.333mL of the glycerol solution and 0.300mL of the calcium chloride solution into 10mL of the silk protein solution after the preparation, and uniformly mixing to obtain a precursor solution 1, wherein the mass fractions of dry substances of the glycerol and the calcium chloride in the precursor solution 1 are respectively 20% and 5%. 2mL of precursor solution 1 (20% glycerol +5% calcium chloride) was poured into 34.8mm circular holes in a 6-hole well plate so that the solution completely covered the bottom surface of the flat bottom of each circular hole. And then, placing the opening of the pore plate poured with the precursor solution 1 in a vacuum drying oven for drying at room temperature for 12 hours to prepare the thin film layer of the silk protein patch.
(2) Preparing a sponge layer: and (3) placing the prepared silk protein patch film layer in a refrigerator at the temperature of-80 ℃ for 2 hours for precooling. 1.429mL of glycerol solution and 0.644mL of calcium chloride solution are added into 10mL of silk protein solution and are uniformly mixed to obtain a precursor solution 2, so that the mass fractions of dry substances of glycerol and calcium chloride in the precursor solution 2 are respectively 20% and 10%. Pouring 2mL of precursor solution 2 (20% of glycerol and 10% of calcium chloride) on the silk fibroin patch film layer, then placing the silk fibroin patch film layer in a refrigerator at minus 80 ℃ for fully freezing for more than 12h, and placing the silk fibroin patch film layer in a freeze dryer for 24h after freezing is finished, thus preparing the silk fibroin double-layer patch with the film and sponge structures.
Fig. 1 is a photograph showing samples prepared in the above preparation steps (1) and (2), illustrating that the film layer and the sponge layer have a significant structural difference.
Fig. 2 shows a cross-sectional scanning electron microscope image of the silk protein double-layer patch prepared as described above, which clearly shows the double-layer structure of the silk protein-based patch, specifically, it shows a spongy structure with a loose and porous upper layer and a thin film structure at the lower layer.
FIG. 3 shows the IR spectrum of the silk protein double-layer patch film layer prepared as above, from which the film can be analyzedFilm layer sample at 1626cm -1 The peak value is higher, the analysis and calculation can show that the sample has higher beta-sheet content which reaches 25 percent, and the skilled person in the art knows that the higher beta-sheet content means that the sample has good mechanical properties.
Test example 1:
the silk fibroin bilayer patch prepared in example 1 was cut into a sheet having a length of 4cm and a width of 2cm, subjected to a stretching test on a universal tester, and the change in stress-strain data was recorded.
Fig. 4 is a tensile test chart of the silk fibroin bi-layer patch film layer prepared as described above, which can be used to obtain a patch having excellent stretchability.
Test example 2:
the silk fibroin double-layer patch prepared in example 1 was cut into a sheet having a length of 4cm and a width of 2cm, two pieces of pigskins having a width of 2cm were bonded together by the patch, an adhesion test was performed on a universal tester, and the change of tension-displacement data was recorded.
Fig. 5 is a graph showing adhesion performance test of the silk protein bi-layer patch prepared as described above, and it can be concluded that the silk protein bi-layer patch adhered to the skin of a pig can produce a significant adhesion force of about 10N/m.
Test example 3:
the silk protein bilayer patch prepared in example 1 was weighed to have an average mass of about 0.24g. It was immersed in 20mL of 0.02mg/mL phosphate buffer. And after fully soaking in a shaking table for 24 hours, taking out the patch, drying, weighing the residual mass, and calculating the dissolving capacity of the patch.
Fig. 6 is a water solubility characterization chart of the silk fibroin bilayer patch prepared as above, which shows that most of the ingredients are dissolved after the patch is soaked in phosphate buffer solution for 24h, and the dissolution percentage is 69.59 +/-7.51%. The mass of the dissolved patch per mL of solution can be calculated to be about 8.44mg.
Test example 4:
mouse fibroblast L929 cells were treated with DMEM +10% FBS at 37 ℃ and 5% CO 2 The cells were cultured routinely (10 cm dishes) when they grew to log phaseCollecting cells, discarding culture solution, washing with PBS for three times, adding 3mL of 0.25% trypsin-0.04% EDTA, digesting at 37 deg.C for 2min, adding 5mL of complete culture medium for neutralization reaction, blowing to remove cells, transferring into a centrifuge tube, centrifuging at 1000rpm for 5min, and adjusting cell suspension concentration to 10 4 one/mL. The cells were plated in 96-well plates, 200. Mu.l of cell suspension was added per well, and the plates were placed in a cell incubator (37 ℃ C., 5% CO) 2 ) And (5) performing conventional culture.
The silk protein bilayer patch prepared in example 1 was immersed in 6.34mL of cell culture medium, and the leachate was collected after sufficient immersion at 37 ℃ for 24 hours. After centrifugation and filtration to ensure sterility, the cells were diluted with DMEM +10% FBS to obtain a series of leachates containing different concentrations of the fibroin-based bilayer patch-dissolving substances (8.44 mg/mL,4.22mg/mL,2.11mg/mL,0.84 mg/mL). Adding the leachate containing the silk protein-based double-layer patch dissolved substances with different concentrations into a culture plate in which mouse fibroblast L929 cells are cultured, and continuously culturing for 24 hours. After 24h 20. Mu.l of CCK8 solution (5 mg/mL) were added and incubation continued for 2h, and the absorbance at 450nm of the well plate was measured with a UV-visible absorptiometer.
Cell viability (i.e., cell viability) was calculated by the following equation to verify the cytocompatibility of the patch in the tissue physiological environment.
Cell viability (%) (%) = [ a (test) -a (blank) ]/[ a (control) -a (blank) ] × 100
A (test): absorbance of wells with cells, CCK8 solution and leachate
A (blank): absorbance of wells with medium and CCK8 solution without cells
A (control): absorbance of wells with cells, CCK8 solution, and no leachate
* Cell viability: cell proliferation Activity or cytotoxic Activity
Fig. 7 shows a cytocompatibility characterization diagram of the silk protein-based bilayer patch prepared in example 1. As can be seen from fig. 7, when the concentration of the silk protein-based double-layer patch-solubilized substance in the leachate was less than or equal to 4.22mg/mL, the cell viability of the mouse fibroblasts showed a level consistent with that of the control group, which can prove that the silk protein-based double-layer patch had good cell compatibility.
Claims (10)
1. A method of preparing a silk protein based bilayer material comprising the steps of:
(1) Respectively preparing silk protein, plasticizer and inorganic salt aqueous solution;
(2) Uniformly mixing the silk protein aqueous solution, the plasticizer aqueous solution and the inorganic salt aqueous solution obtained in the step (1) to obtain a uniform precursor solution 1 of silk protein, plasticizer and inorganic salt;
(3) Preparing the precursor solution 1 into a film form and drying to obtain a film layer;
(4) Uniformly mixing the silk protein aqueous solution, the plasticizer aqueous solution and the inorganic salt aqueous solution obtained in the step (1) to obtain a uniform precursor solution 2 of silk protein, plasticizer and inorganic salt;
(5) And (4) placing the film layer obtained in the step (3) at a temperature of between 20 ℃ below zero and 80 ℃ below zero for precooling, pouring the precursor solution 2 prepared in the step (4) on the frozen film layer, and then carrying out freeze drying to prepare a sponge layer, thereby obtaining the silk protein-based double-layer material with the film and sponge structures.
2. The method according to claim 1, wherein, in step (1), the silk protein aqueous solution is prepared according to the following method:
adding the crushed silkworm cocoons into a boiling sodium carbonate solution, keeping the silkworm cocoons in the boiling sodium carbonate solution for degumming, rinsing and drying the degummed silk, adding the dried degummed silk into a lithium bromide aqueous solution for dissolving, and dialyzing and purifying the solution after all silk fibers are dissolved to obtain a silk protein aqueous solution.
3. The method of claim 2, wherein,
in the preparation of silk protein water solution, the concentration of sodium carbonate solution is 0.01-0.05mol/L, and the degumming treatment time is 5-180 minutes; the concentration of the lithium bromide aqueous solution was 9.3mol/L and dissolution was continued at 60 ℃ for 4 hours.
4. The method according to claim 1, wherein, in step (1),
the plasticizer is selected from glycerol, sorbitol and polyethylene glycol, and the concentration of the water solution of the plasticizer is 50-1000mg/mL;
the inorganic salt is selected from calcium chloride, lithium chloride and lithium bromide, and the concentration of the aqueous solution is 0.01-10mol/L.
5. The method as claimed in claim 1, wherein, in the step (3), the precursor solution 1 prepared in the step (2) is transferred to a pre-fabricated mold to be made in the form of a thin film, and dried at a temperature of 10-80 ℃ for 12-48 hours to prepare a thin film layer having a thickness ranging from 30 to 100 μm.
6. The method of claim 1, wherein,
in the film layer, the mass fraction of the dry matter of the plasticizer is 10-50%, and the mass fraction of the dry matter of the inorganic salt is 2-20%;
in the sponge layer, the mass fraction of the dry matter of the plasticizer is 10-50%, and the mass fraction of the dry matter of the inorganic salt is 2-20%.
7. The method according to claim 1, wherein in the step (5), in the freeze-drying, the film layer after casting the precursor solution 2 is frozen at 20 to-80 ℃ and then dried in a freeze-drying machine to prepare the sponge layer with a thickness ranging from 50 micrometers to 150 micrometers.
8. A silk protein-based bilayer material prepared by the method of any one of claims 1 to 7.
9. Use of the silk protein-based bilayer material of claim 8 in the preparation of a medical material.
10. Use according to claim 9, wherein the medical material is a medical adhesive patch.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2012095731A (en) * | 2010-10-29 | 2012-05-24 | Gunze Ltd | Bioabsorbable medical material |
| US20150045887A1 (en) * | 2012-03-22 | 2015-02-12 | Trb Chemedica International S.A. | Method for repair of ligament or tendon |
| US20170304491A1 (en) * | 2014-09-26 | 2017-10-26 | Sewoncellontech Co., Ltd. | Matrix for restoring soft tissue and producing method therefor |
| CN105524472A (en) * | 2015-12-22 | 2016-04-27 | 傅泽红 | Keratin/silk fibroin blended membrane and preparation method thereof |
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| CN113527895A (en) * | 2021-08-04 | 2021-10-22 | 西湖大学 | Degradable super-tough silk protein-based material and preparation method and application thereof |
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