A multifunctional double-layer 3D printing hydrogel and preparation method thereof

Abstract

The present invention relates to a multifunctional double-layer 3D printing hydrogel and a preparation method thereof. The multifunctional double-layer 3D printing hydrogel comprises an upper layer and a lower layer, wherein the upper layer is a SFMA / MBG hydrogel sheet, and the upper layer is formed by 3D printing of SFMA / MBG bio-ink; the lower layer is a SFMA / BM@M6 hydrogel sheet, and the lower layer is formed by 3D printing of SFMA / BM@M6 bio-ink. Among them, the SFMA / MBG hydrogel sheet of the upper layer is loaded with bioactive glass nanoparticles (MBG), which can effectively promote skull repair and regeneration; the SFMA / BM@M6 hydrogel sheet of the lower layer is loaded with biomimetic cell vesicles (BM@M6), which can release cytokines to the injured part for a long time, and promote the repair of brain injury. The multifunctional double-layer 3D printing hydrogel of the present invention is used for the treatment of traumatic brain injury, effectively solves the problem of skull defect caused by decompressive craniectomy, and continuously and efficiently delivers drugs to the brain parenchyma while solving the problem, so as to achieve effective inhibition of brain inflammation, angiogenesis and neuronal repair.

Description

Technical Field

[0001] The present invention relates to the technical field of medical biomaterials, and in particular to a multifunctional double-layer 3D printing hydrogel and a preparation method thereof. Background Art

[0002] Traumatic brain injury (TBI) is a growing health problem and one of the leading causes of high morbidity and mortality in young people worldwide. Immediately after injury, the blood-brain barrier (BBB) ​​collapses and a neuroinflammatory cascade that triggers brain degeneration is activated. Common pathological consequences of TBI include hematoma, subarachnoid hemorrhage, neuroinflammation, or axonal injury. Most of these brain injuries also lead to long-term physical, emotional, and cognitive consequences. However, despite the significant threat to public health, there is no effective drug treatment for TBI. Clinically, in order to address the increased intracranial pressure caused by conditions such as ischemia and necrosis of brain tissue, decompressive craniectomy is usually used to relieve brain herniation and save the patient's life. Traditional decompressive craniectomy therapy will cause corresponding vacancies in the brain and skull defects after the removal of the bone flap. In addition, traditional drug treatments cannot effectively deliver cytokines from the periphery to the site of injury through the blood-brain barrier. Summary of the invention

[0003] Based on this, the purpose of the present invention is to provide a multifunctional double-layer 3D printed hydrogel and a preparation method thereof. The multifunctional double-layer 3D printed hydrogel described in the present invention is used to treat traumatic brain injury, effectively solves the problem of skull defects caused by decompressive craniectomy, and while solving the problem, continuously and efficiently delivers drugs to the brain parenchyma, achieving effective inhibition of brain inflammation, angiogenesis and neuronal repair.

[0004] A multifunctional double-layer 3D-printed hydrogel comprises an upper layer and a lower layer, wherein the upper layer is a SFMA / MBG hydrogel sheet, which is formed by 3D printing of SFMA / MBG bio-ink; and the lower layer is a SFMA / BM@M6 hydrogel sheet, which is formed by 3D printing of SFMA / BM@M6 bio-ink.

[0005] The present invention uses 3D printing technology to print a multifunctional double-layer 3D printed hydrogel sheet that has both osteogenesis and promotes brain injury repair. In clinical practice, decompressive craniectomy is usually used to treat increased intracranial pressure after brain injury. After decompressive craniectomy, the hydrogel sheet is placed in the position of the bone flap defect. Among them, the upper layer is a SFMA / MBG hydrogel sheet, and the upper SFMA / MBG hydrogel sheet is loaded with bioactive glass nanoparticles (MBG), which can effectively promote skull repair and regeneration; the lower layer is a SFMA / BM@M6 hydrogel sheet, and the lower SFMA / BM@M6 hydrogel sheet is loaded with bionic cell vesicles (BM@M6), which can release cytokines to the injured site for a long time and promote the repair of brain injury. Different from traditional bionic cell vesicles, the bionic cell vesicles (BM@M6) encapsulated in the hydrogel can stay in situ for a long time.

[0006] The present invention aims at the problem that the traditional decompressive craniectomy therapy will cause the corresponding vacancy in the brain and the skull defect after the removal of the bone flap, and designs a 3D printed hydrogel sheet for dual repair of brain tissue and skull, that is, the multifunctional double-layer 3D printed hydrogel described in the present invention, to fill the vacancy after the removal of the bone flap. In traumatic brain injury (TBI), mechanical damage to the central nervous system (CNS) can cause local inflammatory response and recruit immune cells after injury. In order to solve the problem of inflammation after brain injury, the present invention selects cytokines macrophage colony stimulating factor (M-CSF) and interleukin-6 (IL-6) with immunomodulatory effects. M-CSF and IL-6 are conducive to the generation of tissue repair macrophages in the injured area, thereby promoting the resolution of inflammation, angiogenesis and neuronal repair. However, due to the presence of damaged BBB, it has become a huge challenge to deliver drugs from the periphery to the damaged area. The use of nanoparticle technology can bring new opportunities for treating damaged brains. Nanoparticles are used as carrier transporters for drug treatment of various diseases. Biomimetic nanoparticles represent a paradigm shift in nanoparticle design, enabling nanoparticles and organisms to interact and effectively influence the behavior of complex biological systems. The present invention uses biomimetic nanoparticle microglial (BV2) membrane vesicles loaded with M-CSF and IL-6 to prepare biomimetic nanoparticles BM@M6 that can efficiently deliver drugs in the brain.

[0007] Furthermore, the SFMA / MBG bio-ink is prepared by dissolving SFMA in a PBS solution containing LAP and tartrazine and blending the solution with MBG.

[0008] Furthermore, the SFMA / MBG bio-ink is prepared by dissolving 10% SFMA by mass in a PBS solution containing 0.035% LAP and 0.018% tartrazine, and blending the mixture with 10% MBG by mass.

[0009] Furthermore, the SFMA / BM@M6 bio-ink is prepared by dissolving SFMA in a PBS solution containing LAP and tartrazine, blending the solution with BM@M6 and subjecting the solution to ultrasonic treatment.

[0010] Furthermore, the SFMA / BM@M6 bio-ink is prepared by dissolving 15% SFMA by mass in a PBS solution containing 0.025% LAP and 0.05% tartrazine, blending with BM@M6 and ultrasonically treating the mixture.

[0011] A method for preparing a multifunctional double-layer 3D printing hydrogel comprises the following steps:

[0012] Step (a), synthesis of methacrylated silk fibroin (SFMA): synthesizing SFMA;

[0013] Step (b), synthesis of mesoporous bioactive glass (MBG): synthesizing MBG;

[0014] Step (c), preparation of BM drug-loaded vesicles (BM@M6): preparing BM@M6;

[0015] Step (d), preparation of double-layer 3D printed hydrogel scaffold:

[0016] (1) Preparation of SFMA / MBG bio-ink: SFMA was dissolved in a PBS solution containing LAP and tartrazine, and mixed with MBG to obtain SFMA / MBG bio-ink;

[0017] (2) Preparation of SFMA / BM@M6 bio-ink: SFMA was dissolved in a PBS solution containing LAP and tartrazine, and blended with BM@M6 and sonicated to obtain SFMA / BM@M6 bio-ink;

[0018] (3) Using a photocurable biological 3D printer to 3D print the material: import the mesh model, set the printing parameters, add SFMA / MBG bio-ink into the material tank and start printing to form an SFMA / MBG hydrogel sheet; replace the bio-ink in the material tank with SFMA / BM@M6 bio-ink and continue printing to obtain a SFMA / MBG+SFMA / BM@M6 double-layer hydrogel scaffold, which is any of the multifunctional double-layer 3D printed hydrogels described above.

[0019] The present invention prints a double-layer hydrogel sheet through 3D printing technology, the upper layer is a SFMA / MBG hydrogel sheet with bone repair effect, and the lower layer is a SFMA / BM@M6 grid-shaped hydrogel sheet that promotes brain repair. The preparation method of the multifunctional double-layer 3D printed hydrogel described in the present invention specifically involves the synthesis of methacrylated silk fibroin (SFMA), the preparation of microglial vesicles, i.e., the preparation of BM drug-loaded vesicles (BM@M6), the synthesis of mesoporous bioactive glass (BMG), the preparation of methacrylated silk fibroin blended mesoporous bioactive glass 3D printed hydrogel, and the preparation of methacrylated silk fibroin (SFMA) blended BM drug-loaded vesicles (BM@M6) 3D printed hydrogel.

[0020] Furthermore, in step (a), the method for synthesizing methacrylated silk fibroin (SFMA) comprises: removing silkworm pupae from cocoons under Na 2 CO 3 The solution is boiled twice and washed with distilled water to obtain degummed silk fibroin (SF); the degummed silk fibroin (SF) is dried; the dried degummed silk fibroin (SF) is dissolved in a lithium bromide solution, and after the degummed silk fibroin (SF) is completely dissolved, concentrated hydrochloric acid is added, and the solution is centrifuged and filtered; glycidyl methacrylate is added to the filtrate and mixed; the dialysis bag is dialyzed in distilled water for 3-5 days to remove salt, and lyophilized to obtain SFMA. Silk fibroin (SF) is a natural fibrous protein produced by silkworms. It has been used in a variety of biomedical and biotechnological applications, including wound dressings, enzyme fixation matrices, vascular prostheses, and structural implants. It is considered to be a biocompatible material in regenerative medicine applications, has no obvious long-term inflammatory response, and is conducive to tissue repair. The present invention modifies silk fibroin (SF) with methacrylate, so that methacrylated silk fibroin (SFMA) is successfully photo-crosslinked and polymerized.

[0021] Furthermore, in step (b), the method for synthesizing MBG comprises: dissolving CTAB in deionized water and stirring; after CTAB is completely dissolved, adding EA and stirring to form a microemulsion; adding NH 4 OH solution and stirring; slowly adding TEOS, TEP, and CN into the solution; vigorously stirring, the solution gradually becomes turbid to form a white precipitate. The precipitate is washed with EtOH and deionized water respectively, and dried, roasted, and organic matter and nitrate are removed to obtain MBG. The method described in the present invention synthesizes mesoporous bioactive glass nanoparticles (MBG) with excellent osteogenic function.

[0022] Furthermore, in step (c), the method for preparing BM@M6 includes: mixing the cell membrane suspension with the M-CSF solution and the IL-6 solution and ultrasonicating to obtain a BM / M6 mixed solution; passing the BM / M6 mixed solution through polycarbonate porous membranes with pore sizes of 800nm, 400nm, 200nm, and 100nm in sequence, and co-extruding them several times to prepare BM@M6; washing the solution with deionized water to remove free M6, and retaining the supernatant for later use; finally precipitating BM@M6, adding a small amount of PBS solution to mix well, and storing it for later use.

[0023] Furthermore, in step (d), the preparation of SFMA / MBG bio-ink is as follows: 10% SFMA by mass is dissolved in a PBS solution containing 0.035% LAP and 0.018% tartrazine, and mixed with 10% MBG by mass to obtain SFMA / MBG bio-ink.

[0024] Furthermore, in step (d), the preparation of SFMA / BM@M6 bio-ink is as follows: SFMA with a mass fraction of 15% is dissolved in a PBS solution containing 0.025% LAP and 0.05% tartrazine, mixed with BM@M6 and sonicated to obtain SFMA / BM@M6 bio-ink.

[0025] Furthermore, the set printing parameters include peeling distance, peeling speed, peeling recovery speed, lifting height, and lifting speed.

[0026] Furthermore, the set printing parameters also include light intensity, exposure time, number of base layers and base layer exposure time.

[0027] For better understanding and implementation, the present invention is described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the infrared spectrum of methacrylated silk fibroin;

[0029] Figure 2 This is the SEM image of the 3D printed SFMA;

[0030] Figure 3 TEM image of mesoporous bioactive glass nanoparticles;

[0031] Figure 4 This is a TEM image of a cell membrane vesicle;

[0032] Figure 5 These are the brain repair images on the 3rd and 7th days of treatment;

[0033] Figure 6 This is a diagram of skull defect repair. DETAILED DESCRIPTION

[0034] Example 1

[0035] This embodiment provides a method for preparing a multifunctional double-layer 3D printed hydrogel, comprising the following steps:

[0036] Step (a), preparation of methacrylated silk fibroin (SFMA):

[0037] 50 g of silkworm cocoons without pupae were placed in 1 L of 0.05 M Na 2 CO 3 The solution was boiled at 100°C for 30 min twice and washed with distilled water several times to obtain degummed silk fibroin (SF). The degummed silk fibroin (SF) was dried in a ℃ oven. 20 g of the dried degummed silk fibroin (SF) was dissolved in 100 mL of 9.3 M lithium bromide (LiBr) solution (with 0.48 g of NaOH) and stirred at 60°C for 1 hour. After the degummed silk fibroin (SF) was completely dissolved, 1 mL of concentrated hydrochloric acid was added, the solution was centrifuged at 10,000 rpm for 5 min, and then filtered through a miracloth. 12 mL of glycidyl methacrylate was added to the solution and mixed. Stir at 1,000 rpm for 8 h at room temperature. After the reaction was completed, the solution was centrifuged at 10,000 rpm for 5 min, and then dialyzed in distilled water for 3-5 days using a dialysis bag (12-14 kDa) to remove salt. The solution was freeze-dried at -80°C to obtain SFMA. Please refer to Figure 1 In this embodiment, silk fibroin (SF) is modified with methacrylate to obtain methacrylated silk fibroin (SFMA).

[0038] Step (b), preparation of mesoporous bioactive glass nanoparticles (MBG):

[0039] Dissolve 0.7g of hexadecyltrimethylammonium bromide (CTAB) in 33mL of deionized water and stir at 30°C. When the hexadecyltrimethylammonium bromide (CTAB) is completely dissolved, add 10mL of ethyl acetate (EA). After stirring for 30 minutes, microemulsions are formed and 7mL of ammonia water (5mol / L) is added. After stirring for 15 minutes, 3.6mL of tetraethyl orthosilicate (TEOS), 0.36mL of triethyl phosphate (TEP) and 2.277g of calcium nitrate tetrahydrate (CN) are added in sequence every 30 minutes. After strong stirring for 4 hours, the solution gradually becomes opaque and a white precipitate is formed. The white precipitate is collected by centrifugation, washed three times with anhydrous ethanol and deionized water respectively, and dried at 60°C for 24 hours. Finally, the mixture is heated at 600°C in air at 1°C·min -1 The sample was calcined at a heating rate of 5 h to remove organic matter and nitrate, and the mesoporous bioactive glass nanoparticles (MBG) were obtained. Figure 3, mesoporous bioactive glass nanoparticles (MBG), are mesoporous bioactive glass nanoparticles (MBG) with excellent osteogenic function.

[0040] Step (c), preparation of BM drug-loaded vesicles (BM@M6):

[0041] (1) Extract BV2 cell membrane according to the instructions of the membrane protein extraction kit. BV2 cells were provided by Professor Yao Maojin's research group. BV2 cells were incubated in a cell culture dish with a diameter of 10 cm. The cells were cultured in a humid environment containing 5% CO2 at 37°C using DMEM medium containing 10% (v / v) FBS and 1% (v / v) penicillin / streptomycin / amphotericin B solution. When the cells were cultured to about 20-50 million, the cells were collected with a cell scraper and washed twice at 1500 rpm for 5 min in cold PBS (10 mM, pH 7.4). 1 mL of membrane protein extraction reagent A with PMSF added was added to the cells, the cells were fully suspended, and placed in an ice bath for 10-15 min. The sample was repeatedly frozen and thawed twice in liquid nitrogen and room temperature. Centrifuged at 4°C, 3000 rpm for 10 min, and the supernatant was collected. The cell membrane fragments were precipitated by centrifugation at 4000 rpm for 30 min at 4°C, and the supernatant was aspirated to obtain BV2 cell membrane fragments. The cell membrane fragments were lyophilized and stored at -80°C.

[0042] (2) The BV2 cell membrane was dissolved in PBS solution, and repeatedly frozen and thawed twice in liquid nitrogen and room temperature, and ultrasonicated for 5 minutes to obtain a hydrated cell membrane suspension (BM / M6 mixed solution). Using an Avanti micro extruder, the hydrated cell membrane suspension (BM / M6 mixed solution) was sequentially passed through polycarbonate porous membranes with pore sizes of 800nm, 400nm, 200nm, and 100nm, and co-extruded 11 times to prepare BM@M6. The prepared BM@M6 was stored at 4°C for future use. Please refer to Figure 4 , cell membrane vesicles (BM@M6) are biomimetic nanoparticles that can efficiently deliver drugs in the brain.

[0043] Step (d), preparation of double-layer 3D printed hydrogel scaffold:

[0044] (1) Preparation of SFMA / MBG bio-ink: 10% SFMA by mass was dissolved in a PBS solution containing 0.035% LAP and 0.018% tartrazine, and mixed with 10% MBG by mass to obtain SFMA / MBG bio-ink.

[0045] (2) SFMA / BM@M6 bio-ink was prepared by dissolving 15% SFMA by mass in a PBS solution containing 0.025% LAP and 0.05% tartrazine, blending with BM@M6 and ultrasonicating for 1 min to obtain SFMA / BM@M6 bio-ink.

[0046] (3) Use a photocurable bio-3D printer to 3D print the material: First, import the circular grid model and set its diameter to 8 mm and thickness to 2 mm. Then, set the printing parameters, including peeling distance (6 mm), peeling speed (25 mm / min), peeling recovery speed (180 mm / min), lifting height (0 mm), and lifting speed (100 mm / min). In addition, the light intensity (15 mW / cm2), exposure time (15 s), number of base layers (4 layers), and base layer exposure time (50 s) were adjusted. Add SFMA / MBG bio-ink to the material tank and start printing. When 4 layers are printed, pause the printing, replace the bio-ink in the material tank with SFMA / BM@M6 bio-ink and continue printing to obtain the SFMA / MBG+SFMA / BM@M6 double-layer hydrogel scaffold, which is a multifunctional double-layer 3D printed hydrogel.

[0047] Example 2

[0048] A multifunctional double-layer 3D-printed hydrogel in this embodiment is a multifunctional double-layer 3D-printed hydrogel prepared in Example 1, including an upper layer and a lower layer, the upper layer is a SFMA / MBG hydrogel sheet, and the upper layer is 3D-printed from SFMA / MBG bio-ink; the lower layer is a SFMA / BM@M6 grid hydrogel sheet, and the lower layer is 3D-printed from SFMA / BM@M6 bio-ink.

[0049] Example 3

[0050] This example uses the multifunctional double-layer 3D printed hydrogel prepared in Example 1 for the treatment of traumatic brain injury. Decompressive craniectomy is clinically used to treat increased intracranial pressure after brain injury. After decompressive craniectomy, the hydrogel sheet is placed at the location of the craniectomy defect. Figure 5 and Figure 6 Compared with the use of SFMA / MBG+SFMA for the treatment of traumatic brain injury, the SFMA / MBG+SFMA / BM@M6 double-layer hydrogel scaffold, that is, the multifunctional double-layer 3D printed hydrogel, effectively solves the problem of skull defects caused by decompressive craniectomy, and while solving this problem, it continuously and efficiently delivers drugs to the brain parenchyma, achieving effective inhibition of brain inflammation, vascular regeneration and neuronal repair.

[0051] The upper SFMA / MBG hydrogel sheet is loaded with bioactive glass nanoparticles (MBG), which can effectively promote skull repair and regeneration; the lower SFMA / BM@M6 hydrogel sheet is loaded with bionic cell vesicles (BM@M6), which can release cytokines to the injured area for a long time and promote the repair of brain injury. Unlike traditional bionic cell vesicles, the bionic cell vesicles (BM@M6) encapsulated in the hydrogel can stay in situ for a long time.

[0052] Compared with the prior art, the multifunctional double-layer 3D printed hydrogel of the present invention fills the gap left after the clinical use of craniectomy decompression surgery, and at the same time realizes the multifunctional repair of brain tissue and skull.

[0053] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, and the present invention is also intended to include these modifications and modifications.

Claims

1. A multifunctional double-layer 3D printed hydrogel, Features: It comprises an upper layer and a lower layer, wherein the upper layer is a SFMA / MBG hydrogel sheet, and the upper layer is formed by 3D printing of SFMA / MBG bio-ink; the lower layer is a SFMA / BM@M6 hydrogel sheet, and the lower layer is formed by 3D printing of SFMA / BM@M6 bio-ink; The method for preparing the multifunctional double-layer 3D printing hydrogel comprises the following steps: Step (a), SFMA synthesis: Remove the silkworm cocoons from the pupae and place them in Na 2 CO 3 The solution is boiled twice and washed with distilled water to obtain degummed silk fibroin; the degummed silk fibroin is dried; the dried degummed silk fibroin is dissolved in a lithium bromide solution, and after the degummed silk fibroin is completely dissolved, concentrated hydrochloric acid is added, and the solution is centrifuged and filtered; glycidyl methacrylate is added to the filtrate, and mixed; the filtration is dialyzed in distilled water for 3-5 days with a dialysis bag to remove salt, and freeze-dried to obtain SFMA; Among them, SFMA represents methacrylated silk fibroin; Step (b), MBG synthesis: Dissolve CTAB in deionized water and stir. After CTAB is completely dissolved, add EA and stir to form a microemulsion. Add NH 4 OH solution and stir; slowly add TEOS, TEP, and CN into the solution; vigorously stir, the solution gradually becomes turbid to form a white precipitate; wash the precipitate with EtOH and deionized water respectively, and dry, roast, remove organic matter and nitrate to obtain MBG; Wherein, MBG represents mesoporous bioactive glass nanoparticles, CTAB represents hexadecyltrimethylammonium bromide, EA represents ethyl acetate, TEOS represents tetraethyl orthosilicate, TEP represents triethyl phosphate, and CN represents calcium nitrate tetrahydrate; Step (c), preparation of BM@M6: The cell membrane suspension was mixed with the M-CSF solution and the IL-6 solution and ultrasonicated to obtain a BM / M6 mixed solution; the BM / M6 mixed solution was sequentially passed through polycarbonate porous membranes with pore sizes of 800 nm, 400 nm, 200 nm, and 100 nm, and co-extruded 11 times to prepare BM@M6; the solution was washed with deionized water to remove free M6, and the supernatant was reserved for later use; the final precipitation was BM@M6, a small amount of PBS solution was added to mix well, and stored for later use; Among them, BM@M6 represents BM drug-loaded vesicles, BM represents cell membrane, M6 represents M-CSF and IL-6; M-CSF represents macrophage colony-stimulating factor; IL-6 represents interleukin-6; Step (d), preparation of double-layer 3D printed hydrogel scaffold: (1) Preparation of SFMA / MBG bio-ink: SFMA was dissolved in a PBS solution containing LAP and tartrazine, and mixed with MBG to obtain SFMA / MBG bio-ink; (2) Preparation of SFMA / BM@M6 bio-ink: SFMA was dissolved in a PBS solution containing LAP and tartrazine, and blended with BM@M6 and sonicated to obtain SFMA / BM@M6 bio-ink; (3) 3D printing the material using a photocurable biological 3D printer: importing a mesh model, setting printing parameters, adding SFMA / MBG bio-ink into the material tank and starting printing to form an SFMA / MBG hydrogel sheet; replacing the bio-ink in the material tank with SFMA / BM@M6 bio-ink and continuing printing to obtain a SFMA / MBG+SFMA / BM@M6 double-layer hydrogel scaffold, i.e., the multifunctional double-layer 3D printed hydrogel; Here, LAP represents lithium phenyl-2,4,6-trimethylbenzoylphosphite.

2. The multifunctional double-layer 3D printed hydrogel according to claim 1, Features: The SFMA / MBG bio-ink is prepared by dissolving 10% SFMA by mass in a PBS solution containing 0.035% LAP and 0.018% tartrazine, and blending the mixture with 10% MBG by mass.

3. The multifunctional double-layer 3D printing hydrogel according to claim 1, Features: The SFMA / BM@M6 bio-ink is prepared by dissolving 15% SFMA by mass in a PBS solution containing 0.025% LAP and 0.05% tartrazine, and blending with BM@M6 and ultrasonically treating the mixture.

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