A silk fibroin composite chitosan-based scaffold material with a directional porous structure and a preparation method thereof

Abstract

The application belongs to the technical field of artificial biological materials, and particularly relates to a silk fibroin composite chitosan-based scaffold material with a directional porous structure and a preparation method thereof. Silk fibroin, polyethylene glycol and chitosan are mixed in a solution form in a mass ratio of 1:1-1.5:1-5, the obtained mixed solution is poured into a mold composed of an aluminum plate as a heat-conducting bottom plate and heat-insulating plates in other parts, the mold is placed in a low-temperature environment for directional freezing, the frozen silk fibroin-chitosan composite material is demolded and transferred to a vacuum freeze dryer for sublimation drying, so that the silk fibroin composite chitosan-based scaffold material with the directional porous structure is obtained.

Description

Technical Field

[0001] This invention belongs to the field of artificial biomaterials technology, and specifically relates to a silk fibroin composite chitosan-based scaffold material with a directional porous structure and its preparation method. Background Technology

[0002] Bone loss caused by trauma or surgery is called a bone defect. Because of bone defects, nonunion, slow or non-union, and local functional impairment are common problems. Millions of people worldwide develop bone defects each year due to severe trauma, fractures complicated by infection, unhealed fractures, bone tumors, or other comorbidities. Treatment for bone defects is lengthy and the outcome is uncertain, causing immense physical and psychological suffering for patients. Traditional treatments like bone transplantation have drawbacks such as immune rejection and donor shortages, while current methods like implanting metal scaffolds face issues like biocompatibility. Therefore, finding a suitable material for repairing bone defects is crucial.

[0003] Chitosan (CS) is a natural polymer extracted from the shells of crustaceans. Firstly, compared to synthetic materials, chitosan has excellent hydrophilicity, rapid biodegradation, and the resulting chitosan oligosaccharides after depolymerization impart good antibacterial properties. Its monomeric products can also be metabolized by the human body or directly excreted. Secondly, as a bone scaffold material, chitosan can promote osteoconduction and osteogenic mineralization in vivo. Furthermore, chitosan is beneficial for wound healing, hemostasis, and anti-inflammatory effects. These advantages make chitosan a preferred material for bone defect repair. However, traditional chitosan-based scaffold materials often lack the complex structure and properties of natural bone tissue. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for preparing a silk fibroin composite chitosan-based scaffold material with a directional porous structure.

[0005] Silk fibroin, polyethylene glycol, and chitosan were mixed thoroughly in solution at a mass ratio of 1:1 to 1.5:1 to 5. The resulting mixed solution was then subjected to directional freeze-drying to obtain a silk fibroin composite chitosan-based scaffold material with a directional porous structure.

[0006] As a preferred option, silk fibroin is obtained by fully dissolving natural silk in an aqueous lithium bromide solution after degumming, followed by dialysis.

[0007] Preferably, polyethylene glycol is dissolved in acetic acid solution to obtain polyethylene glycol solution, and then mixed thoroughly with silk fibroin solution to obtain silk fibroin / polyethylene glycol solution, and the silk fibroin / polyethylene glycol solution is mixed thoroughly with chitosan solution to obtain mixed solution.

[0008] Furthermore: the acetic acid solution is prepared by thoroughly mixing acetic acid and water at a volume ratio of 2:98, and polyethylene glycol is dissolved in the acetic acid solution at a mass fraction of 2-5%.

[0009] Furthermore: In the silk fibroin solution, the mass fraction of silk fibroin is 2-5%.

[0010] Furthermore, in the chitosan solution, the mass fraction of chitosan is 2-5%.

[0011] As a preferred method, the mixed solution is poured into a mold consisting of a base plate as a heat-conducting substrate and other parts as heat-insulating plates, and the mold is closed and sealed. After the mold is placed in a low-temperature environment for directional freezing, the frozen silk fibroin-chitosan composite material is demolded and transferred to a vacuum freeze dryer for sublimation drying, thereby obtaining a silk fibroin composite chitosan-based scaffold material with a directional porous structure.

[0012] Furthermore: the mold is a hollow columnar structure with a vertical axis and a cross-sectional area of ​​5–15 cm². 2 The height is 0.5 to 2.5 cm.

[0013] Furthermore: the thermal insulation board is a high-density polytetrafluoroethylene board.

[0014] Furthermore: the thermally conductive substrate is a copper plate, gold plate, or aluminum plate, and its thickness is less than 1 mm.

[0015] Furthermore, the low-temperature environment is a liquid nitrogen environment, and the directional freezing time is more than 2 minutes.

[0016] The present invention also provides a silk fibroin composite chitosan-based scaffold material with a directional porous structure obtained by the above preparation method.

[0017] The beneficial effects of this invention are as follows: silk fibroin and chitosan have biodegradability and tissue repair capabilities. By introducing silk fibroin into the interior of chitosan-based bone defect repair material and combining it with directional freeze-drying technology, the porous structure of the scaffold is also oriented, which is conducive to the directional growth of cells, the formation of new tissues and the growth of blood vessels, thereby promoting tissue repair.

[0018] Silk fibroin is a protein with rich biological activity, which effectively promotes cell adhesion and growth, such as cell adhesion, proliferation, and differentiation. In the directional freezing process of this scheme, the major molecular chains in the mixed system tend to align in a directional manner, reducing the entanglement between molecules. Based on this, polyethylene glycol (PEG) molecules are initially added to the mixed system. Firstly, chitosan, composed of glucose units, has a relatively rigid spatial structure, which restricts the positions of amino and hydroxyl functional groups on the chitosan molecule. In contrast, silk fibroin is a protein macromolecule with highly variable structure. This causes the various functional groups contained in silk fibroin, such as hydroxyl and ketone groups, to more readily bind with PEG to form hydrogen bonds and van der Waals forces compared to chitosan (the hydroxyl groups at both ends of the PEG molecule readily form hydrogen bonds with the functional groups on the surface of silk fibroin, and the ether groups in the molecule mainly bind with silk fibroin through other interactions). This allows silk fibroin to bind more readily with PEG. Alcohols combine to form a relatively more stable complex. Simultaneously, due to the strong hydrophilicity of polyethylene glycol (besides the ethylene glycol groups (-CH2CH2OH) at both ends of the molecule, the oxygen atoms of the ether bonds in the polyethylene glycol molecule can form hydrogen bonds with water molecules), it tends to bind with water more readily in the mixed system. This allows silk fibroin, whose entanglement and binding effects with chitosan macromolecules gradually weaken during directional freezing, to move closer to the aqueous phase of the mixed system via polyethylene glycol molecules. After freeze-drying, the silk fibroin becomes more concentrated and closer to the pore walls of the channels formed by the sublimation of the aqueous phase. Therefore, throughout the entire process, silk fibroin migrates to the pore wall surface to a certain extent, resulting in a greater concentration of silk fibroin on the pore walls of the scaffold used for cell adhesion, which is beneficial for further enhancing the bioactivity of the scaffold.

[0019] The prepared scaffold maintains an ideal pore structure and is a scaffold material that combines physicochemical properties and biocompatibility, with broad application prospects. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the experimental preparation apparatus for the silk fibroin composite chitosan-based scaffold material with a directional porous structure according to the present invention. Below the transparent culture dish is a liquid nitrogen container filled with cryogenic liquid nitrogen. The top of the container is sealed by the transparent culture dish, which simultaneously supports the directional freezing mold. The culture dish has perforations to allow the bottom plate of the mold to directly contact the liquid nitrogen in the container below. The lower right corner shows a schematic diagram of the mold in an inclined state with the top unsealed.

[0021] Figure 2 The image shows the axial end face morphology of the silk fibroin composite chitosan-based scaffold material with a directional porous structure prepared in Example 1.

[0022] Figure 3A fluorescence microscope image of a cross section (radial section) of the silk fibroin composite chitosan-based scaffold material with a directional porous structure prepared in Example 1;

[0023] Figure 4 The image shows a fluorescence microscope image of a longitudinal section of the silk fibroin composite chitosan-based scaffold material with a directional porous structure prepared in Example 1. Detailed Implementation

[0024] It should be noted that the directional qualifiers "base plate," "vertical," "horizontal," and "height" used in the description of this application refer to the attached... Figure 1 The directions indicated are merely for the purpose of describing the invention and simplifying the description, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as limiting the present application.

[0025] A method for preparing a silk fibroin composite chitosan-based scaffold material with a directional porous structure:

[0026] Acetic acid and water are mixed thoroughly at a volume ratio of 2:98 to obtain an acetic acid solution. Polyethylene glycol is then dissolved in this acetic acid solution at a mass fraction of 2-5% to obtain a polyethylene glycol solution.

[0027] After degumming, natural silk is fully dissolved in an aqueous lithium bromide solution, followed by dialysis to obtain a silk fibroin solution. Water is then added to dilute the solution to a silk fibroin concentration of 2–5%.

[0028] The silk fibroin, polyethylene glycol (number average molecular weight approximately 2000, the same below), and chitosan (number average molecular weight approximately 50000, the same below) are mixed in a mass ratio of 1:1 to 1.5:1 to 5. First, the polyethylene glycol solution and the silk fibroin solution are thoroughly mixed to obtain a silk fibroin / polyethylene glycol solution. Then, this silk fibroin / polyethylene glycol solution is thoroughly mixed with a chitosan solution of 2-5% by mass. The resulting mixture is poured into a container with a cross-sectional area of ​​5-15 cm², consisting of a base plate of thermally conductive aluminum (0.8 mm thick) and other parts made of thermally insulated high-density polytetrafluoroethylene (PTFE) sheets. 2 The silk fibroin-chitosan composite material is placed in a vertically oriented hollow columnar mold with a height of 0.5-2.5 cm and sealed. The mold is then placed above liquid nitrogen and exposed to the liquid nitrogen for directional freezing for more than 2 minutes. After the frozen silk fibroin-chitosan composite material is demolded, it is transferred to a vacuum freeze dryer for sublimation drying, thereby obtaining a silk fibroin composite chitosan-based scaffold material with a directional porous structure.

[0029] Example 1

[0030] Acetic acid and water were mixed thoroughly at a volume ratio of 2:98 to obtain an acetic acid solution. Polyethylene glycol was then dissolved in this acetic acid solution at a mass fraction of 4% to obtain a polyethylene glycol solution.

[0031] After degumming, natural silk is fully dissolved in a sufficient amount of 0.75% lithium bromide aqueous solution, followed by dialysis to obtain a silk fibroin solution. Water is then added to dilute the solution to a silk fibroin protein concentration of 5%.

[0032] The silk fibroin, polyethylene glycol, and chitosan were mixed in a mass ratio of 1:1.2:4. First, 150g of the polyethylene glycol solution and 100g of the silk fibroin solution described in this embodiment were thoroughly mixed to obtain a silk fibroin / polyethylene glycol solution. Then, this silk fibroin / polyethylene glycol solution was thoroughly mixed with 1000g of a 2% (w / w) chitosan aqueous solution.

[0033] The above-obtained mixed solution was poured into a mold cavity with a cross-sectional area of ​​12 cm², consisting of a base plate made of thermally conductive aluminum plate (0.8 mm thick) and other parts made of thermally insulating high-density polytetrafluoroethylene plate. 2 The mold is placed in a hollow columnar structure with a height of 2cm and vertical axis and sealed. The mold is then placed above liquid nitrogen and exposed to the liquid nitrogen downwards for directional freezing for 2 minutes (to ensure sufficient freezing). After the frozen silk fibroin-chitosan composite material is demolded, it is transferred to a vacuum freeze dryer for sublimation drying, thereby obtaining a silk fibroin composite chitosan-based scaffold material with a directional porous structure.

[0034] Example 2

[0035] Acetic acid and water were mixed thoroughly at a volume ratio of 2:98 to obtain an acetic acid solution. Polyethylene glycol was dissolved in this acetic acid solution at a mass fraction of 2% to obtain a polyethylene glycol solution.

[0036] After degumming, natural silk is fully dissolved in a sufficient amount of 0.75% lithium bromide aqueous solution, followed by dialysis to obtain a silk fibroin solution. Water is then added to dilute the solution to a silk fibroin protein concentration of 2%.

[0037] The silk fibroin, polyethylene glycol, and chitosan were mixed in a mass ratio of 1:1:5. First, 100g of the polyethylene glycol solution and 100g of the silk fibroin solution described in this embodiment were thoroughly mixed to obtain a silk fibroin / polyethylene glycol solution. Then, this silk fibroin / polyethylene glycol solution was thoroughly mixed with 200g of a 5% (w / w) chitosan aqueous solution.

[0038] The above-obtained mixed solution was poured into a mold cavity with a cross-sectional area of ​​5 cm², consisting of a base plate made of thermally conductive aluminum plate (0.8 mm thick) and other parts made of thermally insulated high-density polytetrafluoroethylene (PTFE) plate. 2The silk fibroin-chitosan composite material was placed in a vertically oriented hollow columnar mold with a height of 1 cm and sealed. The mold was then placed above liquid nitrogen and brought into contact with the liquid nitrogen for directional freezing for 2 minutes (to ensure sufficient freezing). After the frozen silk fibroin-chitosan composite material was demolded, it was transferred to a vacuum freeze dryer for sublimation drying, thereby obtaining a silk fibroin composite chitosan-based scaffold material with a directional porous structure.

[0039] Comparative Example 1

[0040] Without the addition of polyethylene glycol, the solid content of the mixed solution used in the freezing mold was adjusted to be the same as in Example 1, and the remaining components and operations were the same as in Example 1.

[0041] After degumming, natural silk is fully dissolved in a sufficient amount of 0.75% lithium bromide aqueous solution, followed by dialysis to obtain a silk fibroin solution. Water is then added to dilute the solution to a silk fibroin protein concentration of 5%.

[0042] The silk fibroin solution and chitosan were mixed thoroughly at a mass ratio of 1:4. Then, 508g of water was added and mixed thoroughly.

[0043] The above-obtained mixed solution was poured into a mold cavity with a cross-sectional area of ​​12 cm², consisting of a base plate made of thermally conductive aluminum plate (0.8 mm thick) and other parts made of thermally insulating high-density polytetrafluoroethylene plate. 2 The mold is placed in a hollow columnar structure with a height of 2cm and vertical axis and sealed. The mold is then placed above liquid nitrogen and exposed to the liquid nitrogen downwards for directional freezing for 2 minutes (to ensure sufficient freezing). After the frozen silk fibroin-chitosan composite material is demolded, it is transferred to a vacuum freeze dryer for sublimation drying, thereby obtaining a silk fibroin composite chitosan-based scaffold material with a directional porous structure.

[0044] Compare with Example 1

[0045] Directional freezing was not used; all other components and procedures were the same as in Example 1.

[0046] Acetic acid and water were mixed thoroughly at a volume ratio of 2:98 to obtain an acetic acid solution. Polyethylene glycol was dissolved in this acetic acid solution at a mass fraction of 4% to obtain a polyethylene glycol solution.

[0047] After degumming, natural silk is fully dissolved in a sufficient amount of 0.75% lithium bromide aqueous solution, followed by dialysis to obtain a silk fibroin solution. Water is then added to dilute the solution to a silk fibroin protein concentration of 5%.

[0048] The silk fibroin, polyethylene glycol, and chitosan were mixed in a mass ratio of 1:1.2:4. First, 150g of the polyethylene glycol solution and 100g of the silk fibroin solution from this comparative example were thoroughly mixed to obtain a silk fibroin / polyethylene glycol solution. Then, this silk fibroin / polyethylene glycol solution was thoroughly mixed with 1000g of a 2% (w / w) chitosan aqueous solution.

[0049] The resulting mixture was poured into a mold cavity made entirely of thermally conductive aluminum with a cross-sectional area of ​​12 cm². 2 The silk fibroin-chitosan composite material was placed in a vertically oriented hollow columnar mold with a height of 2cm and sealed. The mold was then immersed in liquid nitrogen for 2 minutes (to ensure complete freezing). After the frozen silk fibroin-chitosan composite material was demolded, it was transferred to a vacuum freeze dryer for sublimation drying, thereby obtaining a silk fibroin composite chitosan-based scaffold material with a directional porous structure.

[0050] Compare with Example 2

[0051] No polyethylene glycol was added, and the solid content of the mixed solution used in the freezing mold was adjusted to be the same as in Control Example 1. All other components and operations were the same as in Control Example 1.

[0052] After degumming, natural silk is fully dissolved in a sufficient amount of 0.75% lithium bromide aqueous solution, followed by dialysis to obtain a silk fibroin solution. Water is then added to dilute the solution to a silk fibroin protein concentration of 5%.

[0053] The silk fibroin solution and chitosan were mixed thoroughly at a mass ratio of 1:4. Then, 508g of water was added and mixed thoroughly.

[0054] The resulting mixture was poured into a mold cavity made entirely of thermally conductive aluminum with a cross-sectional area of ​​12 cm². 2 The silk fibroin-chitosan composite material was placed in a vertically oriented hollow columnar mold with a height of 2cm and sealed. The mold was then immersed in liquid nitrogen for 2 minutes (to ensure complete freezing). After the frozen silk fibroin-chitosan composite material was demolded, it was transferred to a vacuum freeze dryer for sublimation drying, thereby obtaining a silk fibroin composite chitosan-based scaffold material with a directional porous structure.

[0055] Cell adhesion and growth experiments

[0056] The silk fibroin composite chitosan-based scaffold materials with oriented porous structures prepared in the above-described embodiments, comparative embodiments, and control examples were horizontally cut into 1.5 mm thick sheets at the center of the height direction (radial cross-section remained unchanged). The sheets were immersed in a sufficient amount of 75% ethanol aqueous solution for 2 hours, then immersed in a sufficient amount of 0.01M PBS buffer solution for 1 hour. After removing the scaffold material and vacuum drying, it was irradiated with a UV lamp for 45 minutes. The sheets were then further cut to obtain several small circular pieces with a diameter slightly smaller than the pore size of a 96-well plate.

[0057] Small discs of the same sample prepared in the same embodiment were laid out one-to-one in the wells of a 96-well plate and immersed in α-MEM medium. Human umbilical artery smooth muscle cells were collected, digested with trypsin (0.25%, containing phenol red), and the resulting cell suspension was counted at a ratio of 3 × 10⁻⁶ cells / well. 3 Cells were seeded at a density of [number] cells / well in the above-mentioned 96-well plates and placed in a cell culture incubator (MCO-18AIC, Sanyo, Japan). After culturing in this medium for three days, the culture medium was discarded, and 10 μL of LTT detection solution (5 mg / mL) was added. The plates were then incubated in an incubator for 4 hours, after which the culture was terminated. 100 μL of DMSO was added, and the plates were shaken for 10 minutes. The absorbance (OD value) was measured at 450 nm using a microplate continuous wavelength microplate reader; a higher OD value indicates a greater number of cells. In the detection experiment, the sample became almost transparent after this period of soaking, which had no significant effect on the absorbance measurement. The results are shown in the table below.

[0058] OD value Example 1 1.7 Example 2 1.4 Comparative Example 1 0.9 Compare with Example 1 0.7 Compare with Example 2 0.9

[0059] In the table above, in Comparative Example 1, polyethylene glycol was not used as a "hydrophilic guide" compared to Example 1. Therefore, in Comparative Example 1, silk fibroin molecules could not be targeted to migrate closer to the aqueous phase region in the mixed system during the directional freezing process. As a result, there was relatively less silk fibroin distributed near the pore walls in the scaffold after freeze-drying, which is used for cell adhesion and proliferation, resulting in weaker overall biological activity of the scaffold.

[0060] In the control examples where directional freeze-drying was not used, the cell activity of the scaffold prepared in Control Example 1, after the introduction of polyethylene glycol, was actually worse than that in Control Example 2, which did not include polyethylene glycol. This is because in conventional freezing processes, macromolecular segments do not exhibit a directional effect. Therefore, in both Control Examples 1 and 2, chitosan molecules consistently exhibited significant entanglement and binding effects on silk fibroin. Consequently, although polyethylene glycol, which is compatible with the aqueous phase, was introduced in Control Example 1, it failed to effectively guide the silk fibroin to the aqueous phase region of the mixed system. At the same time, because the proportion of silk fibroin decreased (under the same solid content) after the addition of a certain amount of polyethylene glycol in Control Example 1, the overall cell activity of the scaffold actually decreased.

[0061] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a silk fibroin composite chitosan-based scaffold material with a directional porous structure, characterized in that: The preparation method is as follows: polyethylene glycol is dissolved in acetic acid solution to obtain polyethylene glycol solution, and then mixed thoroughly with the silk fibroin solution to obtain silk fibroin / polyethylene glycol solution. The silk fibroin / polyethylene glycol solution is mixed thoroughly with the chitosan solution to obtain mixed solution. Silk fibroin, polyethylene glycol and chitosan are mixed thoroughly in solution at a mass ratio of 1:1 to 1.5:1 to 5. The resulting mixed solution is then subjected to directional freeze-drying to obtain silk fibroin composite chitosan-based scaffold material with directional porous structure.

2. The method for preparing the silk fibroin composite chitosan-based scaffold material with a directional porous structure as described in claim 1, characterized in that: The silk fibroin is obtained by degumming natural silk, fully dissolving it in a lithium bromide aqueous solution, and then dialysis.

3. The method for preparing the silk fibroin composite chitosan-based scaffold material with a directional porous structure as described in claim 2, characterized in that: The acetic acid solution is prepared by thoroughly mixing acetic acid and water at a volume ratio of 2:98, and the polyethylene glycol is dissolved in the acetic acid solution at a mass fraction of 2-5%.

4. The method for preparing the silk fibroin composite chitosan-based scaffold material with a directional porous structure as described in claim 1, characterized in that: In the silk fibroin solution, the mass fraction of the silk fibroin is 2-5%.

5. The method for preparing the silk fibroin composite chitosan-based scaffold material with a directional porous structure as described in claim 1, characterized in that: In the chitosan solution, the mass fraction of chitosan is 2-5%.

6. The method for preparing the silk fibroin composite chitosan-based scaffold material with a directional porous structure as described in claim 1, characterized in that: The mixed solution is poured into a mold consisting of a base plate that is a heat-conducting substrate and other parts that are heat-insulating plates, and the mold is closed and sealed. The mold is then placed in a low-temperature environment for directional freezing until it is fully frozen. The frozen silk fibroin-chitosan composite material is then demolded and transferred to a vacuum freeze dryer for sublimation drying, thereby obtaining a silk fibroin composite chitosan-based scaffold material with a directional porous structure.

7. The method for preparing the silk fibroin composite chitosan-based scaffold material with a directional porous structure as described in claim 6, characterized in that: The mold is a hollow columnar structure with a vertical axis and a cross-sectional area of ​​5–15 cm². 2 The height is 0.5 to 2.5 cm, the heat insulation board is a high-density polytetrafluoroethylene board, and the heat-conducting substrate is a copper plate, gold plate or aluminum plate.

8. The method for preparing the silk fibroin composite chitosan-based scaffold material with a directional porous structure as described in claim 6, characterized in that: The low-temperature environment is a liquid nitrogen environment, and the directional freezing time is more than 2 minutes.

9. A silk fibroin composite chitosan-based scaffold material with a directional porous structure obtained by the preparation method according to any one of claims 1 to 8.

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