CN110721342B - Low-crystal silk protein scaffold and preparation method thereof - Google Patents
Low-crystal silk protein scaffold and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a preparation method of a low-crystal silk protein scaffold, which comprises the following steps: uniformly blending silk protein aqueous solution and low-concentration small molecular alcohol aqueous solution to obtain silk protein mixed solution, inducing silk protein molecules to be wound and entangled by using small molecular alcohol, and simultaneously avoiding the silk protein conformation from being transformed by using the low concentration of the small molecular alcohol; then freezing the silk protein mixed solution to obtain a silk protein frozen body, and promoting phase separation by utilizing a freezing and icing process; then unfreezing the fibroin frozen body, and forming a three-dimensional porous scaffold with a uniform structure by using the melting of the ice core; finally, the dry low-crystal silk protein porous scaffold which is easy to store is prepared by a freeze-drying process.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to a low-crystal silk protein scaffold and a preparation method thereof.
Background
Patients with organ or tissue damage and loss of function due to disease and accidents are in the millions each year, requiring over 800 million surgeries annually in the united states alone to treat such patients, with an economic cost of over 4000 billion dollars. With the development of modern medical and surgical techniques, the repair of functional losses by tissue or organ transplantation has become widely accepted, but faces a huge donor gap. The formation of tissues or organs in vivo or in vitro by regenerative medical procedures provides a new treatment regimen for the repair of impaired function. Wherein, the selection and the construction of the tissue engineering scaffold material are one of the keys of the treatment method.
Fibroin is a natural high molecular biomaterial derived from the nature, has excellent mechanical properties, controllable biodegradability and easy processability, and particularly has biocompatibility equal to that of collagen, so that the fibroin is an ideal raw material of a regenerative medical stent. China is the main production country of silk, and the silk yield accounts for more than 70% of the world yield. In recent years, the research and application of silk have been extended from the traditional textile field to the high and new technical field, such as photoelectron and biomedical materials, and especially, the research and application of silk as biomedical materials have made important progress.
The silk fibroin porous scaffold has been widely studied because it has a three-dimensional porous structure, can provide a three-dimensional space for adhesion and proliferation of cells and regeneration of tissues, and is advantageous for the transmission of nutrients. At present, there are many methods for preparing the silk protein porous scaffold, including freeze drying, salting out method, gas foaming method, three-dimensional printing and the like. The silk fibroin mainly has two crystal forms of silk fibroin I (silk I) and silk fibroin II (silk II), which are also the structural basis of natural silk that is insoluble in water and has excellent mechanical properties, however, the crystal forms also hinder the degradation characteristics of silk fibroin. In order to realize water insolubility, the silk protein scaffold prepared at present mainly forms a silk II crystal after crosslinking or post-treatment, and partially forms a silk I crystal structure. However, one problem with current silk protein scaffolds is their high crystallinity resulting in poor degradation. Although amorphous silk protein scaffolds can be prepared, amorphous silk protein scaffolds are readily soluble in water and have no applicability. At present, the silk protein scaffold with the silk I structure can be prepared through water vapor post-treatment, and the degradation performance of the scaffold is improved. However, at present, no report exists on the water-insoluble low-crystal silk protein porous scaffold, and no related preparation method exists. CN200410024711.9 discloses a preparation method of a silk protein three-dimensional porous material, which adopts alcohol with 0.01-20 times of the volume of silk protein solution as a denaturant to induce protein denaturation, and the prepared scaffold mainly adopts silk II crystal.
Therefore, the problems of the existing processing technology and the silk protein scaffold structure are overcome, the low-crystal and water-insoluble silk protein scaffold and the preparation method thereof are developed, and the method has great significance for the application of silk protein in the field of biomedical materials and the clinical application of regenerative medicine.
Disclosure of Invention
The invention aims to provide a novel silk protein scaffold and a preparation method thereof, and solves the problem that the prior art can not prepare a low-crystallinity water-insoluble silk protein scaffold.
The first purpose of the invention is to provide a preparation method of an oligofibroin bracket, which comprises the following steps:
(1) preparing a fresh fibroin aqueous solution according to a conventional method, adjusting the concentration of the solution, and then uniformly mixing the solution with a low-concentration small molecular alcohol aqueous solution to obtain a mixed solution; the small molecular alcohol is one or more of methanol, ethanol, n-propanol, isopropanol and ethylene glycol.
(2) Injecting the mixed solution into a mold for freezing to obtain a mixed solution frozen body;
(3) unfreezing the mixed solution frozen body in air or water bath to obtain a wet low-crystal silk protein scaffold;
(4) fully soaking the wet low-crystal silk protein scaffold in deionized water to remove small molecular alcohol;
(5) freezing the wet silk protein branch scaffold with the alcohol removed again, and then freezing and drying to obtain a dry low-crystal silk protein scaffold;
further, in the step (1), the preparation method of the aqueous solution of silk protein comprises the following steps:
degumming silk, dissolving with neutral salt, and dialyzing to obtain silk protein water solution. Preferably, the silk is mulberry silk.
Further, in the step (1), the concentration of the silk protein aqueous solution is 0.1-10 wt%. Preferably, the concentration of the silk protein aqueous solution is 1-5 wt%. More preferably, the concentration of the silk protein in the water solution is 2-4 wt%.
Further, in step (1), the silk protein in the aqueous solution of silk protein is in an amorphous structure.
Further, in the step (1), the concentration of the aqueous solution of the small molecular alcohol is 0.01-0.99 wt%. Preferably, the concentration of the aqueous solution of the small molecular alcohol is 0.1-0.5 wt%. More preferably, the concentration of the aqueous solution of the small molecular alcohol is 0.1-0.3 wt%. In the invention, the concentration of the aqueous solution of the small molecular alcohol is lower, and the low-degree aggregation winding of silk protein molecules can be induced by reducing the concentration of the alcohol, and meanwhile, the transition of the conformation of the silk protein molecules from amorphous state to beta-folding is avoided, and the silk I or silk II crystal is formed. If the concentration of the small molecular alcohol is higher than the range of the application, beta-folding is easily generated, and further crystallization is generated to form a high-crystallization silk protein scaffold.
Further, in the step (1), the volume ratio of the silk protein aqueous solution to the small molecular alcohol aqueous solution is 1-50: 1 to 50. Preferably, the volume ratio of the silk protein aqueous solution to the small molecular alcohol aqueous solution is 1-5: 1 to 5.
Preferably, in step (1), the small molecule alcohol is ethanol.
Further, in the steps (2) and (4), the freezing temperature is-4-80 ℃. More preferably, the freezing temperature is from-20 ℃ to 60 ℃.
Further, in the step (2), the freezing treatment time is 1-24 h. More preferably, the freezing treatment time is 6-12 h.
Further, in the step (3), the thawing temperature is 0-60 ℃. More preferably, the thawing temperature is 20 ℃ to 37 ℃.
The principle of the method of the invention is as follows: the invention selects amorphous silk protein aqueous solution, adds low-concentration alcohol solution into the solution, induces silk protein molecules to generate low-degree aggregation winding through alcohol molecules of organic solvent, and simultaneously avoids the conversion and crystallization of silk protein molecule conformation to beta-folding. Then, the silk protein molecules are frozen by a freeze-drying process, and phase separation is promoted by using ice nuclei formed by water, wherein the silk protein molecules are aggregated to form porous pore walls. And finally, unfreezing to melt off ice nuclei to form the low-crystal silk protein scaffold.
The second purpose of the invention is to provide the low-crystal silk protein scaffold prepared by the preparation method.
Further, silk protein in the scaffold is mainly in an amorphous structure, and the crystallinity is lower than 30%.
The low-concentration micromolecular alcohol adopted by the invention has two remarkable characteristics: firstly, silk protein molecules are promoted to be wound and entangled, and a water-insoluble silk protein scaffold can be directly formed after being frozen; secondly, the low concentration of the small molecular alcohol is utilized to avoid the silk protein molecular conformation from being converted into a beta-folding structure till crystallization, and the novel low-crystallization silk protein scaffold is obtained. The preparation process only needs to add trace small molecular alcohol, is easy to remove, and has mild preparation process conditions, simple process, convenient operation and easy quantification. The silk protein scaffold prepared by the method has the characteristics of complete and uniform porous structure, higher porosity, good plasticity and low crystallinity, and is a silk protein scaffold material with novel structural characteristics.
By the scheme, the invention at least has the following advantages:
the method has the advantages of mild conditions, simple process, convenient operation and easy quantification. The invention prepares a novel silk protein scaffold with low crystallinity for the first time, and the scaffold is insoluble in water. The low crystallinity is beneficial to regulating and controlling the degradation performance of the silk protein. Therefore, the silk protein scaffold prepared by the invention is mainly of a three-dimensional porous structure, the secondary structure is mainly of an amorphous structure, the scaffold has low crystallinity, high hydrophilicity, good hot water stability and controllable degradation performance, is very beneficial to the delivery of nutrient substances, the migration of cells and the growth of tissues, and meanwhile, the degradation of materials can be matched with the growth of the tissues, so that the scaffold is an ideal tissue engineering scaffold and has wide application in the fields of regenerative medicine, drug loading and the like.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.
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FIG. 1 shows the appearance (A), scanning electron micrograph (B), infrared spectrum (C) and X-ray diffraction pattern (D) of the fibroin scaffold prepared in the first example.
FIG. 2 shows an IR spectrum (A) and an X-ray diffraction pattern (B) of the silk protein scaffold prepared in the comparative example.
Detailed Description
The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
In the following examples, unless otherwise specified, "concentration" is a mass percentage concentration.
Example one
About 10g of raw silk was dipped in 0.5L of 0.5% Na2CO3And stirring and boiling the solution for 60 minutes, taking out the solution, and washing the solution clean by deionized water. Repeating the above operations twice, and drying the silk at 60 ℃.
5g of the degummed silk after the treatment is weighed and dissolved in 30mL of LiBr solution with the concentration of 9.8mol/L, and the solution is stirred and dissolved for one hour at the temperature of 60 ℃. Then dialyzing with deionized water for 3 days by using a dialysis bag with molecular weight cutoff of 3500 to obtain a silk protein aqueous solution with the concentration of 6%, and further adjusting the concentration of the silk protein aqueous solution to 2%.
And taking 10mL of the fibroin solution, adding 5mL of glycol solution with the mass fraction of 0.5%, uniformly stirring, pouring into a polyethylene mould, and then putting the mould containing the mixed solution into a mould at-20 ℃ for freezing for 6 hours. And then taking out the frozen body, unfreezing the frozen body in deionized water for 1h, rinsing the frozen body with the deionized water for three times to obtain an insoluble silk protein scaffold, and freeze-drying the insoluble silk protein scaffold again to obtain the low-crystal silk protein porous scaffold.
The results of electron microscope scanning and infrared testing of the above fibroin scaffolds are shown in FIG. 1. As can be seen from FIG. 1B, the inside of the silk protein scaffold is mainly porous structure, and in addition, the infrared absorption peak of the silk protein is located at 1640cm-1This is the characteristic peak of the amorphous structure (fig. 1C). The X-ray diffraction curve of silk protein showed no crystalline diffraction peaks (fig. 1D), thereby confirming that the silk protein scaffold prepared in this example has a mainly amorphous structure, and the degree of crystallinity was calculated to be 12% and the hot water dissolution rate was less than 2%. The silk protein scaffold prepared in this example was therefore of low crystalline structure.
Example two
About 50g of raw silk was immersed in 2L of 0.05% Na2CO3And stirring and boiling the solution for 60 minutes, taking out the solution, and washing the solution clean by deionized water. Repeating the above operations twice, and drying the silk at 60 ℃.
Weighing 15g of the degummed silk after the treatment, dissolving the degummed silk in 100mL of LiBr solution with the concentration of 9.8mol/L, and stirring and dissolving the degummed silk for one hour at 60 ℃. Then dialyzing with deionized water for four days by using a dialysis bag with the molecular weight cutoff of 3500 to obtain a fibroin aqueous solution with the concentration of 7 percent, and adjusting the concentration of the fibroin aqueous solution to 4 percent.
Taking 15mL of the fibroin solution, uniformly mixing the fibroin solution with 15mL of ethanol with the mass fraction of 0.1%, then pouring the mixture into a polyethylene mould, and then putting the polyethylene mould into a place to be frozen for 10 hours at the temperature of minus 20 ℃. And (3) taking out after freezing, unfreezing in air for 2h at natural temperature, rinsing with deionized water for 3h, and finally freezing and drying to obtain the low-crystal silk protein scaffold.
The structure of the silk protein scaffold is tested, and the crystallinity is calculated to be 10 percent, and the hot water dissolution loss rate is lower than 3 percent.
EXAMPLE III
About 50g of raw silk was immersed in 2L of 0.05% Na2CO3And stirring and boiling the solution for 60 minutes, taking out the solution, and washing the solution clean by deionized water. Repeating the above operations twice, and drying the silk at 60 ℃.
Weighing 15g of the degummed silk after the treatment, dissolving the degummed silk in 100mL of LiBr solution with the concentration of 9.8mol/L, and stirring and dissolving the degummed silk for one hour at 60 ℃. Then dialyzing with deionized water for four days by using a dialysis bag with the molecular weight cutoff of 3500 to obtain a fibroin aqueous solution with the concentration of 7 percent, and adjusting the concentration of the fibroin aqueous solution to 5 percent.
Taking 15mL of the fibroin solution, uniformly mixing the fibroin solution with 30mL of methanol with the mass fraction of 0.01 percent, pouring the mixture into a polyethylene mould, and then putting the polyethylene mould into a place to be frozen for 12 hours at the temperature of minus 20 ℃. And (3) taking out after freezing, unfreezing in air for 1h at natural temperature, rinsing with deionized water for 3h, and finally freezing and drying to obtain the low-crystal silk protein scaffold.
Example four
About 50g of raw silk was immersed in 5L of 0.05% Na2CO3The solution is stirred and boiled for 30 minutes, then taken out and washed clean by deionized water. Repeating the above operations for three times, and naturally drying the silk.
Weighing 15g of the degummed silk after the treatment, dissolving the degummed silk in 30mL of LiBr solution with the concentration of 9.8mol/L, and stirring and dissolving the degummed silk for 3 hours at 60 ℃. Then dialyzing with deionized water for 3 days by using a dialysis bag with the molecular weight cutoff of 3500 to obtain a silk protein aqueous solution with the concentration of 6 percent, and adjusting the concentration of the silk protein aqueous solution to 3 percent.
And taking 10mL of the fibroin solution, adding 2mL of isopropanol solution with the mass fraction of 0.8%, uniformly stirring, pouring into a polyethylene mould, and then putting into a polyethylene mould to be frozen for 4 hours at the temperature of minus 40 ℃. And then unfreezing the mixture in deionized water for 2 hours, rinsing the mixture for three times by using the deionized water to obtain an insoluble silk protein scaffold, and freeze-drying the insoluble silk protein scaffold again to obtain the low-crystal silk protein porous scaffold.
EXAMPLE five
About 50g of raw silk was immersed in 2L of 0.5% Na2CO3And stirring and boiling the solution for 60 minutes, taking out, washing the solution with deionized water, and naturally drying the solution.
Weighing 15g of the degummed silk after the treatment, dissolving the degummed silk in 100mL of LiBr solution with the concentration of 9.8mol/L, and stirring and dissolving the degummed silk for one hour at 60 ℃. Then dialyzing with deionized water for four days by using a dialysis bag with the molecular weight cutoff of 3500 to obtain a silk protein aqueous solution with the concentration of 5 percent, and adjusting the concentration of the silk protein aqueous solution to 4 percent.
Taking 15mL of the fibroin solution, uniformly mixing the fibroin solution with 15mL of ethanol with the mass fraction of 0.5%, then pouring the mixture into a polyethylene mould, and then putting the polyethylene mould into a place to be frozen for 6 hours at the temperature of minus 20 ℃. And taking out after freezing, directly soaking in deionized water for thawing for 1h, rinsing with deionized water for 2h, and finally freezing and drying to obtain the low-crystal silk protein scaffold.
EXAMPLE six
About 50g of raw silk was immersed in 5L of 0.2% Na2CO3Stirring and boiling the solution for 60 minutes, taking out the solution, and using deionized water
Washing with water, and drying at 60 deg.C.
Weighing 15g of the degummed silk after the treatment, dissolving the degummed silk in 100mL of LiBr solution with the concentration of 9.8mol/L, and stirring and dissolving the degummed silk for one hour at 60 ℃. Then dialyzing with deionized water for four days by using a dialysis bag with the molecular weight cutoff of 3500 to obtain a silk protein aqueous solution with the concentration of 6 percent.
Taking 15mL of the fibroin solution, uniformly blending the fibroin solution with 60mL of n-propanol with the mass fraction of 0.5%, then pouring the mixture into a polyethylene mould, and then putting the polyethylene mould into a mould to be frozen for 12 hours at the temperature of minus 80 ℃. And (3) taking out after freezing, unfreezing in air for 4h at natural temperature, rinsing with deionized water for 3h, and finally freezing and drying to obtain the low-crystal silk protein scaffold.
Comparative example
About 10g of raw silk was dipped in 0.5L of 0.5% Na2CO3And stirring and boiling the solution for 60 minutes, taking out the solution, and washing the solution clean by deionized water. Repeating the above operations twice, and drying the silk at 60 ℃.
5g of the degummed silk after the treatment is weighed and dissolved in 30mL of LiBr solution with the concentration of 9.8mol/L, and the solution is stirred and dissolved for one hour at the temperature of 60 ℃. Then dialyzing with deionized water for 3 days by using a dialysis bag with molecular weight cutoff of 3500 to obtain a silk protein aqueous solution with the concentration of 6%, and further adjusting the concentration of the silk protein aqueous solution to 2%.
And taking 10mL of the fibroin solution, adding 5mL of glycol solution with the mass fraction of 10%, uniformly stirring, pouring into a polyethylene mould, and then putting the mould containing the mixed solution into a mould at the temperature of minus 20 ℃ for freezing for 8 hours. And then taking out the frozen body, unfreezing the frozen body in deionized water for 1h, rinsing the frozen body with the deionized water for three times to obtain an insoluble silk protein scaffold, and freeze-drying the insoluble silk protein scaffold again to obtain the silk protein porous scaffold.
The results of the infrared test on the above silk protein scaffold are shown in FIG. 2. As can be seen from FIG. 2A, the infrared absorption peak of silk protein is located at 1626cm-1This is the characteristic peak of the beta-sheet conformation. As can be seen from FIG. 2B, the X-ray diffraction pattern of silk protein shows a plurality of crystal diffraction peaks at 9.6, 20.5 and 24.6 degrees, respectively. Thus, the silk protein scaffold prepared by the comparative example is determined to be mainly of a beta-sheet crystal structure, and the crystallinity is 45% and the hot water dissolution loss rate is lower than 1%. The silk protein scaffold prepared in this comparative example was mainly of a high crystalline structure.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Claims (8)
1. The preparation method of the low-crystal silk protein scaffold is characterized by comprising the following steps of:
(1) uniformly mixing the aqueous solution of silk fibroin and the aqueous solution of micromolecular alcohol to obtain a mixed solution; the micromolecular alcohol is one or more of methanol, ethanol, n-propanol, isopropanol and glycol; the concentration of the aqueous solution of the small molecular alcohol is 0.1-0.99 wt%;
(2) injecting the mixed solution into a mold for freezing to obtain a mixed solution frozen body;
(3) unfreezing the mixed solution frozen body to obtain a wet-state low-crystal silk protein scaffold;
(4) removing the small molecular alcohol in the wet low-crystal silk protein scaffold, and then freeze-drying to obtain the dry low-crystal silk protein scaffold.
2. The method of claim 1, wherein: in the step (1), the silk protein in the silk protein water solution is mulberry silk.
3. The method of claim 1, wherein: in the step (1), the concentration of the silk fibroin aqueous solution is 0.1-10 wt%; the volume ratio of the silk protein aqueous solution to the small molecular alcohol aqueous solution is 1-50: 1 to 50.
4. The method of claim 1, wherein: in the steps (2) and (4), the freezing temperature is-4-80 ℃, and the freezing treatment time is 1-24 h.
5. The method of claim 1, wherein: in the step (3), the thawing temperature is 0-60 ℃.
6. The method of claim 1, wherein: in the step (4), the wet-state low-crystal silk protein scaffold is soaked in water to remove the small molecular alcohol in the wet-state low-crystal silk protein scaffold.
7. An oligofibroin scaffold prepared by the method of any one of claims 1 to 6.
8. The oligofilament protein scaffold according to claim 7, wherein: the silk fibroin in the scaffold is mainly in an amorphous structure, and the crystallinity is lower than 30%.
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