A method for preparing a porous silk fibroin bone cement scaffold by using low-temperature 3D printing technology

Abstract

The application discloses a method for preparing a porous silk fibroin bone cement scaffold by using a low-temperature 3D printing technology, and belongs to the technical field of biomedical materials. The method comprises the following steps: adopting a coaxial nozzle to extrude shell ink containing silk fibroin, alginate and bioactive ceramics and a sacrificial core ink to a low-temperature platform with a gradient distribution of temperature; after the formed frozen core-shell structure silk bundle is subjected to freeze drying, the silk bundle is immersed into a calcium ion solution, so that the elution and removal of the sacrificial core are realized synchronously to construct mesopores, and the ion crosslinking of the alginate is realized to enhance the structure; and finally, the final solidification is completed through mild ethanol vapor treatment. The application can construct a three-level biomimetic pore structure with macropores, mesopores and gradient micropores, and the prepared scaffold has excellent structure controllability and mechanical properties.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a method for preparing a bone repair scaffold, and particularly a method for constructing a silk fibroin-based composite bone cement scaffold with multi-level channels and a biomimetic structure using low-temperature 3D printing technology. Background Technology

[0002] Tissue engineering technology offers an important approach to bone defect repair, among which low-temperature 3D printing technology based on silk fibroin composites has attracted much attention due to its customizability and good biocompatibility. This technology can construct bone repair scaffolds with porous structures with high fidelity by extruding silk fibroin-based bio-ink at low temperatures and freezing it immediately.

[0003] However, existing technologies still face three major bottlenecks in practical applications. First, the pore structure of the prepared scaffolds is too simple, typically consisting only of macropores formed by printing and micropores formed by freeze-drying, lacking the intermediate-scale channels crucial for effective cell migration and vascularization. Second, the inherent contradiction between high porosity and mechanical strength is difficult to reconcile, making it difficult for the scaffolds to provide long-term effective mechanical support in load-bearing areas. Finally, the commonly used liquid-phase alcohol treatment process, while inducing stabilization of silk fibroin, often irreversibly damages its delicate microporous structure.

[0004] Therefore, developing a new method that can construct biomimetic multi-level channels and synergistically solve the problems of mechanical performance and structural stability is a key technology that urgently needs to be broken through in this field. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of the prior art by providing a method for fabricating porous silk fibroin bone cement scaffolds using low-temperature 3D printing technology. This method can construct scaffolds with a three-level biomimetic pore structure of "macropore-medium pore-micro-controlled pores," effectively solving the technical problems of traditional scaffolds having a single, uncontrollable pore structure and insufficient mechanical properties.

[0006] The above-mentioned technical objective of this invention is achieved through the following technical solution: a method for preparing porous silk fibroin bone cement scaffolds using low-temperature 3D printing technology, comprising the following steps:

[0007] a) Provide shell inks containing silk fibroin and alginate, and removable sacrificial core inks;

[0008] b) The shell ink and core ink are extruded together through a coaxial nozzle to a low-temperature platform with a temperature gradient to form a frozen core-shell structured filament bundle;

[0009] c) Freeze-dry the frozen core-shell structured filament bundle to form micropores in the shell layer of the filament bundle;

[0010] d) The freeze-dried core-shell structured filament bundles were immersed in a solution containing calcium ions to simultaneously achieve the elution and removal of the core layer and the ionic crosslinking of alginate;

[0011] e) Apply ethanol vapor to the ion-crosslinked core-shell structured fibrous bundles to induce β-sheet formation and solidify the silk fibroin.

[0012] Furthermore, the temperature gradient distribution in step b) causes the aperture of the microcontroller formed in step c) to vary gradient along a specific direction of the filament bundle.

[0013] Furthermore, in step d), the core layer is eluted and removed, forming a through-channel structure inside the filament bundle.

[0014] Furthermore, the shell ink also includes bioactive ceramics, which are composed of a group consisting of bioactive glass and hydroxyapatite.

[0015] Furthermore, the temperature range of the cryogenic platform is between -15°C and -50°C.

[0016] Furthermore, the temperature gradient is generated by regulating multiple thermoelectric refrigeration units deployed at the bottom of the platform.

[0017] Furthermore, the core ink contains Prönnik F-127 or gelatin.

[0018] Furthermore, the ethanol vapor treatment described in step e) is carried out for 6 to 24 hours under non-immersion conditions to prevent the collapse of the microporous structure.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. This invention utilizes a single printing process, employing printing path planning (forming macropores), coaxial extrusion technology (forming mesopores), and gradient freezing technology (forming gradient micropores), to fabricate a biomimetic structure with pores at three scales. This structure highly mimics the structure of natural bone, facilitating cell migration, proliferation, and the exchange of nutrients and metabolic waste, thus reserving channels for the ingrowth of new blood vessels.

[0021] 2. This invention combines "coaxial printing to form hollow filament bundles" with "ionic cross-linking enhancement," which, while ensuring high porosity and internal connectivity, endows the scaffold with initial mechanical strength and structural stability. Furthermore, the hollow structure ensures material transport channels while also conforming to engineering mechanics design, while ionic cross-linking further strengthens the toughness of the filament bundles.

[0022] 3. This invention employs a two-step mild treatment of "ion crosslinking + steam curing" to replace the traditional liquid-phase alcohol treatment. The simultaneous ion crosslinking provides pre-support for the structure, while the subsequent non-immersion steam curing effectively avoids the damage to the fine microporous structure caused by the liquid-solid interface tension. This maximizes the preservation of the micromorphology formed by gradient freezing and helps protect the activity of the preloaded bioactive molecules. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the process flow of the preparation method of the present invention;

[0024] Figure 2 This is a schematic diagram of the low-temperature coaxial 3D printing system in this invention;

[0025] Figure 3 This is a schematic diagram illustrating the microstructural evolution of a single filament bundle in this invention;

[0026] Figure 4 This is a schematic diagram of the multi-level channel structure of the support of the present invention. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to the accompanying drawings and a specific embodiment. It should be noted that the specific embodiment described herein is merely for illustrative purposes and does not constitute any limitation on the scope of the invention.

[0028] Reference Figures 1 to 4 This embodiment provides a complete process for preparing porous silk fibroin bone cement scaffolds using low-temperature 3D printing technology.

[0029] Step 1: Preparation of bio-ink

[0030] The preparation method of the shell layer ink is as follows: Silkworm cocoons are boiled in a 0.5% Na₂CO₃ solution to degummify them. After washing and drying, they are dissolved in a 9.3M lithium bromide solution, followed by dialyzing in deionized water for three days to obtain a purified regenerated silk fibroin solution, the concentration of which is adjusted to 8% (w / v). Bioactive glass powder with an average particle size of 200 nm is added to this solution, making its mass account for 30% of the dry weight of the silk fibroin. Simultaneously, low-viscosity sodium alginate is added, bringing the final concentration in the ink to 1% (w / v). The mixture is then mechanically stirred and ultrasonically dispersed in an ice-water bath until a uniform and stable suspension is formed, which is the shell layer ink. The preparation method of the core layer ink is as follows: Pluronic F-127 powder is dissolved in deionized water at 4°C to prepare a 25% (w / v) solution, which is then stored at low temperature for later use, resulting in the sacrificial core layer ink.

[0031] Step 2: Perform low-temperature coaxial 3D printing

[0032] The prepared shell ink and core ink are loaded into two separate syringes and connected to a coaxial printhead. In this embodiment, the inner needle (for delivering the core ink) of the coaxial printhead has an inner diameter of 200 μm, and the outer needle (for delivering the shell ink) has an inner diameter of 500 μm. The extrusion rate of the core ink is set to 1 mL / h, and the extrusion rate of the shell ink is set to 3 mL / h. Printing is performed on a custom-designed cryogenic platform, which, through multiple thermoelectric cooling units arranged at its bottom, forms a temperature gradient on its surface that linearly transitions from -20°C at the edge to -40°C at the center. The printer moves at a speed of 5 mm / s along a preset CAD model path (e.g., a 0° / 90° staggered grid structure), extruding the core-shell composite filament onto the cryogenic platform. During this process, the filament freezes instantly upon contact with the platform surface. The temperature gradient on the printing platform causes differences in the freezing rate of the filament at different locations. In the low-temperature region of -40°C, the extremely rapid freezing rate causes water to form tiny ice crystals; while in the relatively high-temperature region of -20°C, the slower freezing rate allows ice crystals a longer growth time, resulting in larger ice crystals.

[0033] Step 3: Freeze-dry the frozen support.

[0034] The printed cryogenic support was quickly transferred to a freeze dryer and dried for 24 hours at -50°C and a vacuum level below 10 Pa. During the freeze-drying process, the solid ice crystals in the filament shell sublimated directly into water vapor and were removed, leaving an interconnected network of micropores in the locations occupied by the ice crystals. Because the ice crystals formed in the previous step exhibited a gradient size distribution, the pore size of the micropores formed after sublimation also showed a corresponding gradient change.

[0035] Step 4: Perform sacrificial layer elution and ion crosslinking

[0036] The freeze-dried scaffold was completely immersed in a 100 mM calcium chloride (CaCl2) aqueous solution and treated in a constant temperature water bath at 37°C for 2 hours, followed by rinsing several times with deionized water to remove residual ions. In this step, two processes occur simultaneously: firstly, the 37°C warm water environment allows the sacrificial material, Prönnicke F-127, to dissolve and be eluted rapidly, forming continuous hollow channels (i.e., mesopores) with a diameter of approximately 200 μm inside the filament bundle, greatly improving the internal connectivity of the scaffold; secondly, calcium ions in the solution undergo ion exchange reactions with the sodium alginate molecular chains in the shell, forming a physical calcium alginate gel network, which rapidly strengthens and fixes the macroscopic structure of the scaffold.

[0037] Step 5: Perform vapor phase curing treatment

[0038] The scaffold, having undergone the above steps and fully absorbed water, was placed on a grid and suspended inside a sealed container. The bottom of the container contained a 75% (v / v) ethanol-water solution as a vapor source. The scaffold was subjected to a non-immersion steam treatment at room temperature for 12 hours. This step utilizes the gentle inducing effect of ethanol vapor. Ethanol molecules, acting as a dehydrating agent, disrupt the hydration layer surrounding the silk fibroin molecular chains, causing them to transform from a random coil conformation to a thermodynamically more stable β-sheet structure, thus achieving final solidification. Compared to traditional direct immersion, the steam treatment process is more gentle, avoiding drastic solvent exchange and capillary forces, and preserving the fine microstructure formed by freeze-drying without causing structural shrinkage or collapse.

[0039] Through the above steps, the final porous silk fibroin bone cement scaffold is obtained, and its structure is shown in the attached figure. Figure 4 As shown, the pores have three scales: macropores with a diameter of 300-600 μm formed by the stacking of printing paths, mesopores with a diameter of about 200 μm formed inside the filament bundle after the removal of sacrificial ink, and micropores with a diameter between 5-50 μm and a gradient distribution formed on the filament bundle wall.

[0040] The macropores provide a basic framework for the macroscopic ingrowth of tissue cells and the initial construction of vascular networks. Mesopores, approximately 200 μm in diameter, serve as continuous channels penetrating the interior of the filament bundles, mimicking natural bone and providing excellent pathways for rapid cell migration into the scaffold and deep nutrient transport, effectively solving the problem of central region cell necrosis caused by poor internal material exchange in traditional large-pore scaffolds. Micropores, ranging from 5 to 50 μm in diameter and distributed in a gradient, provide an ideal microenvironment for the adhesion, proliferation, and differentiation of individual cells, and promote intercellular signal transduction.

[0041] Furthermore, this invention effectively resolves the inherent contradiction between high porosity and mechanical strength by combining "coaxial printing to form hollow filament bundles" with "ionic cross-linking enhancement." The resulting scaffold possesses the aforementioned biological advantages of high connectivity while also exhibiting sufficient initial mechanical strength and structural stability to meet the load-bearing requirements in the early stages of bone defect repair. Simultaneously, due to the use of a mild vapor curing process, the scaffold's fine microporous structure is completely preserved, avoiding structural collapse caused by traditional processes.

[0042] The above are merely specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing porous silk fibroin bone cement scaffolds using low-temperature 3D printing technology, characterized in that, Includes the following steps: a) Provide shell inks containing silk fibroin and alginate, and removable sacrificial core inks; b) The shell ink and core ink are extruded together through a coaxial nozzle to a low-temperature platform with a temperature gradient to form a frozen core-shell structured filament bundle; c) Freeze-dry the frozen core-shell structured filament bundle to form micropores in the shell layer of the filament bundle; d) The freeze-dried core-shell structured filament bundles were immersed in a solution containing calcium ions to simultaneously achieve the elution and removal of the core layer and the ionic crosslinking of alginate; e) The ion-crosslinked core-shell structured filament bundles are subjected to ethanol vapor treatment to induce β-sheet formation and solidification of the silk fibroin; the ethanol vapor treatment lasts for 6 to 24 hours and is carried out under non-immersion conditions to prevent the collapse of the microporous structure. The temperature range of the cryogenic platform is between -15°C and -50°C. The core ink contains either Prönnicke F-127 or gelatin.

2. The method of claim 1, wherein: The temperature gradient distribution in step b) causes the micropores formed in step c) to form on the fiber bundle wall, and the pore size is gradient-distributed between 5-50 μm.

3. The method of claim 1, wherein: In step d), the core layer is eluted and removed, forming a through-channel structure inside the filament bundle.

4. The method of claim 1, wherein: The shell ink further comprises bioactive ceramics, which are composed of a group consisting of bioactive glass and hydroxyapatite.

5. The method of claim 1, wherein: The temperature gradient is generated by regulating multiple thermoelectric cooling units located at the bottom of the platform.

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