Oriented osteogenesis induction skull patch based on MXene dual-electrode plate and pulsed direct-current electric field and preparation method of oriented osteogenesis induction skull patch based on MXene dual-electrode plate and pulsed direct-current electric field

Abstract

The invention discloses a directional osteogenesis induction skull patch based on an MXene dual-electrode plate and a pulsed direct-current electric field and a preparation method of the directional osteogenesis induction skull patch. The method specifically comprises the following steps: 1) performing intercalation on MXene by using a bacterial cellulose solution as an intercalation agent, and combining a layer-by-layer blade coating and laminating process to obtain a composite MXene electrode plate; (2) preparing a porous GelMA pre-solution loaded with bone marrow mesenchymal stem cells; 3) cutting the composite MXene electrode plate into a proper shape to obtain an upper-layer electrode plate and a lower-layer electrode plate, tiling the lower-layer electrode plate as a substrate, dispensing a cell-loaded porous GelMA pre-solution on the surface of the lower-layer electrode plate, covering the upper-layer electrode plate, and performing gelation by combining lateral UV curing to obtain a double-layer electrode plate composite gel material; and 4) carrying out in-vitro culture on the double-layer electrode plate composite gel material for several days, including electrical stimulation culture and non-electrical stimulation culture, and then stripping the upper electrode plate and the lower electrode plate to obtain the skull patch. According to the skull patch, in-vivo skull defect repair can be achieved, and a brand-new skull regeneration and repair strategy is created.

Description

Technical Field

[0001] The present invention relates to a preparation technology of bone repair materials, and in particular to a directional osteogenic induction skull patch based on a MXene double electrode sheet and a pulsed direct current electric field and a preparation method thereof. Background Art

[0002] Skull defect is a very common neurosurgical disease, which is usually caused by trauma, surgery and other reasons. Skull defect may lead to problems such as skull defect syndrome, brain herniation and neurological localization signs, which seriously affect the patient's life safety. Therefore, the treatment of skull defect has always been a huge problem faced by clinicians.

[0003] At present, the clinical methods for treating skull defects mainly include the use of autologous repair materials or artificial synthetic materials for repair. However, the sources of autologous repair materials are limited, and the biological activity of artificial synthetic materials is poor, so there is a lot of room for improvement in promoting skull regeneration and repair. In recent years, skull repair tissue engineering materials have gradually developed, such as hydrogels and porous scaffold materials, which have achieved certain results in this field. However, many of the materials that have been reported cannot solve the problems of cell sources and directional osteogenic differentiation, resulting in a long skull defect repair cycle and unsatisfactory repair effects in the later stage.

[0004] In response to the above problems, the present invention introduces a novel in situ double-layer electrode sheet structure and pulsed direct current stimulation to achieve directional osteogenic differentiation of the gel patch loaded with BMSCs, which is finally implanted as a skull repair cloth sheet, greatly improving the effect of skull repair and providing a new idea for the subsequent design and development of skull repair materials. Summary of the invention

[0005] The purpose of the present invention is to provide a directional osteogenic induction skull patch based on a MXene dual-electrode sheet and a pulsed direct current electric field and a preparation method thereof, in order to solve the problems of low efficiency of traditional skull defect repair, which greatly improves the effect of skull repair and provides a new idea for the subsequent design and development of skull repair materials.

[0006] The present invention is implemented by the following technical solutions:

[0007] A method for preparing a directional osteogenic induction skull patch based on a MXene double electrode sheet and a pulsed direct current electric field comprises the following steps:

[0008] 1) Using an intercalation agent to intercalate MXene under low temperature and ultrasonic conditions, combined with layer-by-layer scraping and lamination processes and sterilization, to obtain a composite MXene electrode sheet;

[0009] 2) preparing a porous methacryloyl gelatin (GelMA) pre-solution and loading bone marrow mesenchymal stem cells (BMSCs) to form a cell-loaded porous GelMA solution;

[0010] 3) Cutting the composite MXene electrode sheet according to the shape of the skull defect area to obtain two composite MXene electrode sheets, leaving an electrode clamping part at one end of each electrode sheet; under light-proof conditions, laying one electrode sheet flat as a substrate, i.e., a lower electrode sheet, dripping a cell-loaded porous GelMA solution on its surface, and then covering another electrode sheet as an upper electrode sheet, combining lateral UV curing to achieve gelation of the cell-loaded porous GelMA solution, to obtain a double-layer electrode sheet composite gel material, forming a three-layer sandwich structure of a lower electrode sheet-a cell-loaded gel layer-an upper electrode sheet;

[0011] 4) The double-layer electrode sheet composite gel material is cultured in vitro for several days, including electrical stimulation culture and non-electrical stimulation culture, and then the upper and lower electrode sheets are peeled off, and the middle layer is obtained as a skull patch, which can be implanted into the skull defect area for efficient repair.

[0012] Furthermore, during the intercalation treatment in step 1), the intercalation agent is a bacterial cellulose solution with a concentration of 0.25-1.25wt%, the size of MXene is 1-3 microns, the concentration is 0.1-0.75wt%, the low temperature is 4-10°C, the ultrasonic power is 150-200W, and the ultrasonic time is 60-120min.

[0013] Furthermore, in step 1), during the layer-by-layer scraping process, the scraper is 0.5-1 mm away from the surface, and the unidirectional scraping is performed at a rate of 3-10 cm / s;

[0014] During the lamination process, a pressurizing block is used for pressurization, the pressure is 5-10MPa, and the pressurization time is 15-30s;

[0015] The composite MXene electrode sheet has a thickness of 200-800 microns.

[0016] Furthermore, in step 2), during the preparation of the porous GelMA pre-solution, under light-proof and sterile conditions, the GelMA substitution degree is 60-90%, the concentration is 10-17.5wt%, the solvent is PBS solution, the photocuring initiator LAP is added at a concentration of 0.2-0.3wt%, and non-chemically cross-linked gelatin microspheres with a size of 150-250 microns are added at a concentration of 0.5-1.5wt%;

[0017] The number of generations of BMSCs is 2-5, and the concentration is 10 6 -10 7 Pieces / ml.

[0018] Furthermore, in step 3), the shape of the skull defect area is obtained by comparing the patient's CT test results, and the electrode support parts of the two composite MXene electrode sheets are respectively located on different sides of the double-layer electrode sheet composite gel material;

[0019] During the UV curing process, a wavelength of 405 nm, a power of 20-30 W, and a curing time of 20-30 s are used;

[0020] The thickness of the cell-carrying solution after gelation is 0.6 mm-1 cm.

[0021] Furthermore, in step 4), during the electrical stimulation culture process, a pulsed DC electric field is applied by an intelligent electrical stimulation system, the intelligent electrical stimulation system comprising a pulsed DC power supply controlled by an electric field intelligent application program and a sterile wire connected thereto, and a culture mold;

[0022] The sterile wires are respectively connected to the electrode clamping parts on the double-layer electrode sheet composite gel material;

[0023] The culture mold is used for placing the double-layer electrode sheet composite gel material.

[0024] Furthermore, in step 4), the in vitro culture is performed for a total of 14-21 days, wherein the electrical stimulation culture is performed continuously for 3-7 days;

[0025] In the process of applying the pulsed direct current electric field, positive and negative square pulse waves are used, the electric field strength is 100-400mV / mm, the wave width is 1500-2500ms, the cycle is 1000-2000 times / time, and the effect is applied once a day.

[0026] A directional osteogenic induction skull patch based on a MXene double electrode sheet and a pulsed direct current electric field is obtained by using any of the preparation methods described above.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] 1) The present invention uses MXene, bacterial cellulose, porous GelMA, BMSCs, etc. as raw materials, and combines intercalation, layer-by-layer scraping, lamination, cell loading, cutting, drip coating and electrical stimulation to promote osteodifferentiation to prepare a directional osteogenic induction skull patch material based on a MXene double electrode sheet and a pulsed DC electric field. The above raw material selection and process combination are original to the present invention.

[0029] 2) The present invention innovatively develops a strategy for directional induction of osteogenic differentiation of BMSCs by upper and lower dual-electrode pulsed direct current stimulation. By designing a MXene electrode sheet with high biocompatibility and high strength, and by forming a tight sandwich structure in situ between the two electrode sheets and the porous GelMA gel loaded with BMSCs, the electrode sheet can directly and evenly conduct pulsed direct current stimulation signals to the BMSCs inside the gel. Compared with the traditional method of using an electrical stimulation chamber device, the advantages of the present invention are: on the one hand, the electrodes are directly in contact with the cell-carrying gel, the applied voltage is more precise, and the resistance generated in the middle and the consumption of the electric field are smaller; on the other hand, the traditional electric field effect is limited by the direction of the electrodes in the electrical stimulation chamber (the direction of action is horizontal) and the size of the electrical stimulation chamber. Generally, the large distance leads to uneven distribution of the electric field. In the present invention, the electric field is directly applied to both ends of the gel, and the distance between the positive and negative electrodes is only the thickness of the gel layer. The electric field effect is more uniform and the effect is more stable.

[0030] 3) The skull patch in the present invention is composed of a core module consisting of a cell-carrying gel layer and a double-layer electrode sheet, and is combined with a pulsed DC power supply, a sterile wire, a culture mold, and an electric field intelligent application program to assemble an electrical stimulation system suitable for directional osteogenic differentiation. The above system is intelligent, controllable, efficient, and stable, achieving structural innovation and performance improvement over traditional electrical stimulation systems.

[0031] 4) The core module composed of the cell-loaded gel layer + double-layer electrode sheet in the present invention is designed with raw materials and structure in combination with actual needs: porous GelMA is used as the matrix for loading BMSCs, and has a large number of pores inside, which can provide a guarantee for the exchange of nutrients and waste in cell culture; bacterial cellulose is used as an intercalation agent to achieve the intercalation treatment of MXene, and further combined with layer-by-layer scraping and lamination processes to achieve the preparation of high-strength electrode sheets, ensuring the biocompatibility of the electrode sheet and its integrity when torn off after the completion of directed osteogenic differentiation. The most important thing is that the high conductivity of MXene provides a fundamental guarantee for it to serve as an electrode sheet; and the upper and lower electrode sheets and the cell-loaded porous GelMA form a sandwich structure, which makes it possible to efficiently induce directed osteogenic differentiation.

[0032] 5) The MXene electrode sheet in the present invention is prepared by adopting a combined process of intercalation + layer-by-layer scraping + lamination. Through the new application of the old process, the bacterial cellulose is intercalated into the MXene nanosheets, so that the interlayer space is constrained, and further combined with layer-by-layer scraping and lamination, the nanosheets are evenly laid on the substrate, and the structure between the sheets is more compact, thereby obtaining a composite MXene electrode sheet material with high strength and high conductivity; in addition, the combined process of paving + drip coating is used to realize the preparation of the sandwich structure of the skull patch, and the assembly of the core module is realized in situ.

[0033] 6) The present invention combines a novel electrical stimulation directional induction osteogenesis technology, and achieves efficient in vitro osteogenesis induction differentiation of BMSCs by setting specific pulsed DC electric field parameters to prepare a novel skull patch material, and achieves regenerative repair of skull defects with a highly osteogenic cell-loaded gel patch material. The above strategy is innovative. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a schematic diagram of the preparation process of the directional osteogenic induction skull patch based on the MXene double electrode sheet and pulsed DC electric field of the present invention;

[0035] Figure 2 Micro-CT results of in vivo repair of directional osteogenic induced skull patch based on MXene dual electrode sheet and pulsed DC electric field according to an embodiment of the present invention (a corresponds to Example 1, b corresponds to Example 1). DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0037] Comparative Example 1:

[0038] 1) Under light-proof and sterile conditions, a pre-solution containing 0.5 wt% of 200 μm diameter gelatin microspheres and 12 wt% GelMA (substitution degree 60%) was prepared, wherein the solvent was PBS and the photoinitiator was 0.25 wt% dLAP; and the loading generation was 3 generations and the concentration was 10 6 BMSCs / ml formed a cell-laden porous GelMA solution;

[0039] 2) The cell-laden gel pre-solution is poured into a sterile mold and cured by UV (405 nm wavelength, 20 W power, and curing time of 25 s). The thickness of the gel layer is 0.8 mm. A conventional electrical stimulation chamber device (including a cover with insertable electrodes, which is already commercially available; as well as a pulsed DC power supply, sterile wires, and an intelligent electric field application program) is then used to apply a pulsed DC electric field (using positive and negative square pulse waves, an electric field strength of 200 mV / mm, a wave width of 2500 ms, 1000 cycles / times, once a day, for 7 consecutive days) and cultured in vitro for 14 days (electrical stimulation for 7 days, and another 7 days of culture in a supporting culture medium after the end of the electrical stimulation), and can be used to implant into the skull defect area for efficient repair.

[0040] After the cell-laden gel was stimulated by pulsed direct current for 7 days, the live and dead cell fluorescence staining count results showed that the cell survival rate was about 82.6%; it was implanted into a rat skull defect model, and samples were taken 4 weeks later. Micro-CT results showed that the skull repair effect was average, such as Figure 2Middle a.

[0041] Embodiment 1:

[0042] 1) Prepare a 0.25wt% bacterial cellulose solution as an intercalant, use the intercalant to intercalate MXene (concentration of 0.4wt%) with a diameter of about 2-3 microns at 4°C and ultrasonic conditions (ultrasonic power of 150W, ultrasonic time of 60min), combine layer-by-layer scraping (the scraper is 0.5mm away from the surface, and unidirectional scraping is performed at a rate of 5cm / s) and lamination process (pressurizing with a pressure block, the pressure is 7MPa, and the pressing time is 15s) to prepare a composite MXene electrode sheet with a thickness of 500 microns, and irradiate and sterilize for use;

[0043] 2) Under light-proof and sterile conditions, a pre-solution containing 0.5 wt% of 200 μm diameter gelatin microspheres and 12 wt% GelMA (substitution degree 60%) was prepared, wherein the solvent was PBS and the photoinitiator was 0.25 wt% dLAP; and the loading generation was 3 generations and the concentration was 10 6 BMSCs / ml formed a cell-laden porous GelMA solution;

[0044] 3) Cut the MXene electrode sheet into a suitable shape according to the skull defect area, and leave an electrode clamping part at one end; under light-proof conditions, lay the lower electrode sheet flat as a substrate, drip the cell-carrying GelMA pre-solution on its surface, cover the upper electrode sheet (the electrode clamping parts on the two electrode sheets are offset from each other), and combine lateral UV curing (design a hollow circular UV curing device, the size of the internal hollow area is larger than the size of the electrode sheet, and the gel is UV-cured from the side. The curing conditions are: 405nm wavelength, 20W power, and curing time is 25s) to achieve gelation. The thickness of the gel layer is 0.8mm, forming a three-layer sandwich structure of lower electrode sheet-cell-carrying gel layer-upper electrode sheet;

[0045] 4) Connecting an intelligent electrical stimulation system (including a pulsed DC power supply, sterile wires, a culture mold, and an intelligent electric field application program) to the above-mentioned double-layer electrode sheet composite gel material through the electrode clamping part, and applying a pulsed DC electric field (using positive and negative square pulse waves, an electric field strength of 200mV / mm, a wave width of 2500ms, 1000 cycles / times, once a day, and continuous action for 7 days). After 14 days of in vitro culture (electrical stimulation for 7 days, and another 7 days of culture in a supporting culture medium after the end of the electrical stimulation), peel off the upper and lower electrode sheets to obtain the middle layer of skull patch, which can be used to implant into the skull defect area for efficient repair.

[0046] Compared with comparative example 1, this embodiment uses a dual electrode sheet combined with a pulsed DC electric field for directional osteogenic induction. After the cell-loaded gel was subjected to pulsed DC stimulation for 7 days, the live and dead cell fluorescence staining counting results showed that the cell survival rate was about 89.4%. The reason may be that when the dual electrode sheet is used directly to apply electrical stimulation, the distance between the electrode sheets is smaller than that between the metal electrodes on the cover of the plug-in electrode, and the applied voltage is lower, so the damage to the cells is reduced; it was implanted into a rat skull defect model, and samples were taken after 4 weeks. The Micro-CT results showed that the skull repair effect was good, such as Figure 2 Middle b.

[0047] Embodiment 2:

[0048] 1) Prepare a 0.25wt% bacterial cellulose solution as an intercalant, use the intercalant to intercalate MXene (concentration of 0.4wt%) with a diameter of about 2-3 microns at 4°C and ultrasonic conditions (ultrasonic power of 150W, ultrasonic time of 60min), combine layer-by-layer scraping (the scraper is 0.5mm away from the surface, and unidirectional scraping is performed at a rate of 5cm / s) and lamination process (pressurizing with a pressure block, the pressure is 7MPa, and the pressing time is 15s) to prepare a composite MXene electrode sheet with a thickness of 500 microns, and irradiate and sterilize for use;

[0049] 2) Under light-proof and sterile conditions, a pre-solution containing 0.5 wt% of 200 μm diameter gelatin microspheres and 12 wt% GelMA (substitution degree 60%) was prepared, wherein the solvent was PBS and the photoinitiator was 0.25 wt% dLAP; and the loading generation was 3 generations and the concentration was 10 6 BMSCs / ml formed a cell-laden porous GelMA solution;

[0050] 3) Cut the MXene electrode sheet into a suitable shape according to the skull defect area, and leave an electrode clamping part at one end; under light-proof conditions, lay the lower electrode sheet flat as a substrate, drip the cell-loaded porous GelMA solution on its surface, cover the upper electrode sheet (the electrode clamping parts on the two electrode sheets are offset from each other), and combine lateral UV curing (design a hollow circular UV curing device, the size of the internal hollow area is larger than the size of the electrode sheet, and the gel is UV-cured from the side. The curing conditions are: 405nm wavelength, 20W power, and curing time of 25s) to achieve gelation. The thickness of the gel layer is 0.8mm, forming a three-layer sandwich structure of lower electrode sheet-cell-loaded gel layer-upper electrode sheet;

[0051] 4) Connecting an intelligent electrical stimulation system (including a pulsed DC power supply, sterile wires, a culture mold, and an intelligent electric field application program) to the above-mentioned double-layer electrode sheet composite gel material through the electrode clamping part, and applying a pulsed DC electric field (using positive and negative square pulse waves, an electric field strength of 400mV / mm, a wave width of 2500ms, 1000 cycles / times, once a day, and continuous action for 7 days). After 14 days of in vitro culture (electrical stimulation for 7 days, and another 7 days of culture in a supporting culture medium after the end of the electrical stimulation), peel off the upper and lower electrode sheets to obtain the middle layer of skull patch, which can be used to implant into the skull defect area for efficient repair.

[0052] Compared with Example 1, the pulsed DC electric field strength is increased in this example. After the cell-loaded gel was subjected to pulsed DC stimulation for 7 days, the live and dead cell fluorescence staining counting results showed that the cell survival rate was about 87.1%, which may be due to the increased damage to the cells after increasing the electric field strength; it was implanted into a rat skull defect model, and samples were taken after 4 weeks. The Micro-CT results showed that the skull repair effect was improved compared with the example, indicating that the high field strength further improved the effect of inducing cell directional osteogenic differentiation, thereby optimizing the skull defect repair results.

[0053] Embodiment 3:

[0054] 1) Prepare a 0.25wt% bacterial cellulose solution as an intercalant, use the intercalant to intercalate MXene (concentration of 0.4wt%) with a diameter of about 2-3 microns at 4°C and ultrasonic conditions (ultrasonic power of 150W, ultrasonic time of 60min), combine layer-by-layer scraping (the scraper is 0.5mm away from the surface, and unidirectional scraping is performed at a rate of 5cm / s) and lamination process (pressurizing with a pressure block, the pressure is 7MPa, and the pressing time is 15s) to prepare a composite MXene electrode sheet with a thickness of 500 microns, and irradiate and sterilize for use;

[0055] 2) Under light-proof and sterile conditions, a 12 wt% GelMA (substitution degree 60%) pre-solution was prepared, wherein the solvent was PBS and the photoinitiator was 0.25 wt% d LAP; and the loading generation was 3 generations and the concentration was 10 6 BMSCs / ml formed a cell-laden GelMA solution;

[0056] 3) Cut the MXene electrode sheet into a suitable shape according to the skull defect area, and leave an electrode clamping part at one end; under light-proof conditions, lay the lower electrode sheet flat as a substrate, drip the cell-carrying GelMA solution on its surface, cover the upper electrode sheet (the electrode clamping parts on the two electrode sheets are offset from each other), and combine lateral UV curing (design a hollow circular UV curing device, the size of the internal hollow area is larger than the size of the electrode sheet, and the gel is UV-cured from the side. The curing conditions are: 405nm wavelength, 20W power, and curing time is 25s) to achieve gelation. The thickness of the gel layer is 0.8mm, forming a three-layer sandwich structure of lower electrode sheet-cell-carrying gel layer-upper electrode sheet;

[0057] 4) Connecting an intelligent electrical stimulation system (including a pulsed DC power supply, sterile wires, a culture mold, and an intelligent electric field application program) to the above-mentioned double-layer electrode sheet composite gel material through the electrode clamping part, and applying a pulsed DC electric field (using positive and negative square pulse waves, an electric field strength of 200mV / mm, a wave width of 2500ms, 1000 cycles / times, once a day, and continuous action for 7 days). After 14 days of in vitro culture (electrical stimulation for 7 days, and another 7 days of culture in a supporting culture medium after the end of the electrical stimulation), peel off the upper and lower electrode sheets to obtain the middle layer of skull patch, which can be used to implant into the skull defect area for efficient repair.

[0058] Compared with Comparative Example 1, gelatin microspheres were not added in this embodiment, that is, a porous pore structure was not introduced into the gel layer. After the cell-loaded gel was subjected to pulsed direct current stimulation for 7 days, the live and dead cell fluorescence staining counting results showed that the cell survival rate was about 71.3%. The failure to introduce a pore structure inside the gel resulted in a decrease in the exchange efficiency of cell nutrition, oxygen and waste inside the gel. In order to extend the gel degradation cycle and enhance the gel strength, the concentration of GelMA was set to 12wt%. Under this condition, it was difficult for the cell survival rate of direct three-dimensional cell culture to have a good performance; it was implanted into a rat skull defect model, and samples were taken after 4 weeks. The Micro-CT results showed that the skull repair effect was significantly reduced compared with Example 1.

Claims

1. A method for preparing a directional osteogenic induction skull patch based on a MXene double electrode sheet and a pulsed direct current electric field, characterized in that: The following steps are involved: 1) Using an intercalation agent to intercalate MXene under low temperature and ultrasonic conditions, combined with layer-by-layer scraping and lamination processes, to obtain a composite MXene electrode sheet; 2) preparing a porous methacryloyl gelatin (GelMA) pre-solution and loading bone marrow mesenchymal stem cells (BMSCs) to form a cell-loaded porous GelMA solution; 3) Cutting the composite MXene electrode sheet according to the shape of the skull defect area to obtain two composite MXene electrode sheets, leaving an electrode clamping portion at one end of each electrode sheet; under light-proof conditions, laying one electrode sheet flat as a substrate, i.e., a lower electrode sheet, dripping a cell-loaded porous GelMA solution on its surface, and then covering another electrode sheet as an upper electrode sheet, combining lateral UV curing to achieve gelation of the cell-loaded porous GelMA solution, to obtain a double-layer electrode sheet composite gel material; 4) Cultivating the double-layer electrode sheet composite gel material in vitro, including electrical stimulation cultivation and non-electrical stimulation cultivation, and then peeling off the upper and lower electrode sheets to obtain a skull patch.

2. The method for preparing a directional osteogenic induction skull patch based on a MXene double electrode sheet and a pulsed direct current electric field according to claim 1, characterized in that: Step 1) During the intercalation treatment, the intercalant is a bacterial cellulose solution with a concentration of 0.25-1.25wt%, the size of MXene is 1-3 microns, the concentration is 0.1-0.75wt%, the low temperature is 4-10°C, the ultrasonic power is 150-200W, and the ultrasonic time is 60-120min.

3. The method for preparing a directional osteogenic induction skull patch based on a MXene double electrode sheet and a pulsed direct current electric field according to claim 1, characterized in that: In step 1), during the layer-by-layer scraping process, the distance between the scraper and the surface is 0.5-1 mm, and the unidirectional scraping is performed at a rate of 3-10 cm / s; During the lamination process, a pressurizing block is used for pressurization, the pressure is 5-10MPa, and the pressurization time is 15-30s; The composite MXene electrode sheet has a thickness of 200-800 microns.

4. The method for preparing a directional osteogenic induction skull patch based on a MXene double electrode sheet and a pulsed direct current electric field according to claim 1, characterized in that: Step 2) In the process of preparing the porous GelMA pre-solution, under light-proof and sterile conditions, the GelMA substitution degree is 60-90%, the concentration is 10-17.5wt%, the solvent is PBS solution, the photocuring initiator LAP is added at a concentration of 0.2-0.3wt%, and non-chemically cross-linked gelatin microspheres with a size of 150-250 microns are added at a concentration of 0.5-1.5wt%; The number of generations of BMSCs is 2-5, and the concentration is 10 6 -10 7 Pieces / ml.

5. The method for preparing a directional osteogenic induction skull patch based on a MXene double electrode sheet and a pulsed direct current electric field according to claim 1, characterized in that: In step 3), the shape of the skull defect area is obtained by comparing the patient's CT test results, and the electrode support parts of the two composite MXene electrode sheets are respectively located on different sides of the double-layer electrode sheet composite gel material; During the UV curing process, a wavelength of 405 nm, a power of 20-30 W, and a curing time of 20-30 s are used; The thickness of the cell-laden porous GelMA solution after gelation is 0.6 mm-1 cm.

6. The method for preparing a directional osteogenic induction skull patch based on a MXene double electrode sheet and a pulsed direct current electric field according to claim 1, characterized in that: In step 4), during the electrical stimulation culture process, a pulsed DC electric field is applied by an intelligent electrical stimulation system, the intelligent electrical stimulation system includes a pulsed DC power supply controlled by an electric field intelligent application program and a sterile wire connected thereto, and a culture mold; The sterile wires are respectively connected to the electrode clamping parts on the double-layer electrode sheet composite gel material; The culture mold is used for placing the double-layer electrode sheet composite gel material.

7. The method for preparing a directional osteogenic induction skull patch based on a MXene double electrode sheet and a pulsed direct current electric field according to claim 6, characterized in that: In step 4), the in vitro culture is performed for a total of 14-21 days, wherein the electrical stimulation culture is performed continuously for 3-7 days; In the process of applying the pulsed direct current electric field, positive and negative square pulse waves are used, the electric field strength is 100-400mV / mm, the wave width is 1500-2500ms, the cycle is 1000-2000 times / time, and the effect is applied once a day.

8. A directional osteoinductive skull patch based on a MXene dual electrode sheet and a pulsed DC electric field, characterized in that: The method is adopted as described in any one of claims 1 to 7.

Images