Bone tissue regeneration and repair module based on direct-connection electrode cell-loaded biological material and in-vitro electrical stimulation sterile culture mold and preparation method of bone tissue regeneration and repair module

Abstract

The invention discloses a bone tissue regeneration and repair module based on a direct-connection electrode cell-loaded biological material and an in-vitro electrical stimulation sterile culture mold and a preparation method of the bone tissue regeneration and repair module. The preparation method comprises the following steps: (1) obtaining the size parameters of the initial bone tissue regeneration and repair module and the in-vitro electrical stimulation culture sterile mold as required; (2) preparing an in-vitro electrical stimulation culture sterile mold based on the parameters, and respectively arranging double gates on two sides of the mold for connecting an electrode slice with an electrical stimulation power supply and ensuring an internal sterile environment; the upper end of the mold is open and is provided with a cover plate for separation. And 3) preparing a cell-carrying biological material directly connected with the conductive slice electrode, and externally connecting an electric field applying device through the conductive slice electrode. And 4) applying a bionic pulse direct-current electric field to the bone tissue, and performing in-vitro culture to obtain the bone tissue regeneration and repair module. The in-vitro incubation of the bone tissue regeneration and repair module is realized through the cooperation of the direct-connection electrode cell-loaded biological material and the electrical stimulation culture mold.

Description

Technical Field

[0001] The present invention relates to a preparation technology of bone repair materials, and in particular to a bone tissue regeneration and repair module based on a directly connected electrode-loaded cell biomaterial and an in vitro electrical stimulation sterile culture mold and a preparation method thereof. Background Art

[0002] Bone defect is a very common disease in orthopedics, usually caused by trauma, inflammation, bone disease and other factors. Once a bone defect occurs, the most commonly used treatment methods in clinical practice are autologous bone transplantation and artificial bone filling, but the source of autologous bone is limited and it is easy to cause secondary damage; the current artificial bone products are still in the early stages of development and the products are not fully mature, resulting in unsatisfactory results in bone defect repair.

[0003] With the development of tissue engineering technology, many bone repair materials with high biological activity have been developed. Among them, electrical stimulation technology has gradually attracted the attention of scientists due to its high efficiency and ability to mimic the internal electrophysiological environment of the human body. High-quality bone tissue repair materials prepared by electrical stimulation intervention have also gradually developed. However, the current electrical stimulation equipment and the method of applying electrical stimulation still have many defects. For example, the electrodes of the electrical stimulation device are still in the form of traditional rods or sheets, resulting in poor contact with the material support, and the distance between the electrodes and the material support is too large, resulting in poor electric field application effect. How to transform and upgrade the above-mentioned electrical stimulation device and the method of applying electrical stimulation to achieve efficient application of electrical stimulation technology in the field of bone tissue repair is a major technical problem in this field.

[0004] In view of the above problems, the present invention designs a bone tissue regeneration and repair module based on direct-connected electrode-loaded cell biomaterials and in vitro electrical stimulation sterile culture molds and a preparation method thereof. By developing new conductive thin-sheet electrodes and in vitro electrical stimulation sterile culture molds, the direct effect of pulsed DC electric fields on the surface of bone defect repair materials is achieved, deepening the application of electrical stimulation in the field of bone defects. Summary of the invention

[0005] The purpose of the present invention is to provide a bone tissue regeneration and repair module based on direct-connected electrode-loaded cell biomaterials and in vitro electrical stimulation sterile culture molds and a preparation method thereof to address the problems that the electrodes of the electrical stimulation device are still in the form of traditional rods or sheets, resulting in poor contact with the material support, and the distance between the electrodes and the material support is too large, resulting in poor electric field application effect. The present invention can successfully achieve in vitro incubation of the bone tissue regeneration and repair module through the combination of the above-mentioned direct-connected electrode-loaded cell biomaterials and the electrical stimulation culture mold.

[0006] The present invention is implemented by the following technical solution, and the specific steps include:

[0007] A method for preparing a bone tissue regeneration and repair module based on a directly connected electrode-loaded cell biomaterial and an in vitro electrical stimulation sterile culture mold comprises the following steps:

[0008] 1) obtaining the size parameters of the initial bone tissue regeneration and repair module and the in vitro electrical stimulation culture sterile mold as needed, and casting a mold of the initial bone tissue regeneration and repair module;

[0009] 2) preparing the in vitro electrical stimulation culture sterile mold based on the obtained size parameters, wherein the in vitro electrical stimulation culture sterile mold is a hollow cylinder composed of two concentric circles of different diameters, and arc gates capable of vertical push-pull switches are respectively arranged on both sides of each concentric circle of the mold, and the lower end of the gate adopts an embedded groove design; the top of the mold is open and is provided with a cover plate for separation;

[0010] 3) Prepare cell-carrying biomaterials directly connected to conductive sheet electrodes, connect an electric field application device to the double arc gates through the conductive sheet electrodes, and the double gate design and the embedded groove design at the lower end of the gates can ensure the extension of the soft and thin electrode sheet, but isolate the cell culture mold from the external environment, realize the connection between the electrode sheet and the electric stimulation power supply and ensure the internal sterile environment, together forming an in vitro electric stimulation incubation system for bone tissue regeneration and repair module;

[0011] 4) After 14-21 days of in vitro culture by applying a bionic pulsed direct current electric field, a bone tissue regeneration and repair module is obtained, which can be used for bone defect regeneration and repair after in situ implantation.

[0012] Furthermore, in step 1), the inner diameter of the sterile mold for in vitro electrical stimulation culture is 1-3 cm larger than the maximum linear dimension of the bone tissue regeneration and repair module.

[0013] Further, in step 2), the arc length of the arc gate is 0.5-2 cm, the height is 2-3 cm, the thickness is 0.5-1 mm, the distance between the two gates on the same side is 0.2-0.5 cm, and the connection line of the four gates is the same straight line;

[0014] The internal cross-sectional dimensions of the embedded groove are consistent with the bottom dimensions of the gate, with a depth of 400-1000 microns, and the raw materials are either PET or PMMA;

[0015] The cover plate is an outer cover type.

[0016] Furthermore, in step 3), during the preparation of the cell-carrying biomaterial directly connected to the conductive sheet electrode, the cell-carrying biomaterial is placed on the conductive sheet electrode, and another conductive sheet electrode is laid on the upper surface thereof to obtain the cell-carrying biomaterial directly connected to the conductive sheet electrode.

[0017] Furthermore, in step 3), the conductive sheet electrode is prepared by blending a nano conductive filler and a binder and then using a doctor blade method; the nano conductive filler is one of graphene, MXene, and carbon nanotubes, with a concentration of 0.05-0.25wt%; the binder is one or more of sodium alginate, chitosan, and PLGA, with a concentration of 1-5wt%; the conductive sheet electrode has a thickness of 200-600 microns, a shape consistent with that of the cell-carrying biomaterial, and each sheet has an electrode sheet extension with a width of 0.4-1.8 cm and a length of 0.5-2 cm.

[0018] Further, in step 3), during the preparation of the cell-loaded biomaterial, in situ UV light curing or 3D printing is used to prepare a solution containing cells and gelatin microspheres, wherein the solution contains a photoinitiator, the cells are bone marrow mesenchymal stem cells (BMSCs), the number of generations is 2-5, and the loading concentration is 10 6 -10 7 / ml; the carrier of the solution is one or more of methacryloyl gelatin (GelMA), methacryloyl chitosan (ChiMA), methacryloyl hyaluronic acid (HAMA), and methacryloyl sodium alginate (AlgMA), with a concentration of 5-15wt%; the photoinitiator is one or more of LAP, Irgacure, and photoinitiator 2959, with a concentration of 0.1-0.4wt%; the gelatin microspheres are temperature-sensitive physical gelatin microspheres with a diameter of 50-200 microns and a concentration of 0.5-1.5wt%, which are used to form a porous structure of a cell-carrying biomaterial.

[0019] Furthermore, during the in-situ UV light curing process, the solution containing cells and gelatin microspheres is poured into the mold of the initial bone tissue regeneration and repair module, with a UV wavelength of 405 nm, a power of 10-20 W, and a time of at least 20 seconds.

[0020] Furthermore, in step 4), the bionic pulsed DC electric field action parameters applied include: voltage 3-8V, positive and negative pulse square wave, wave width 1500-2500ms, wave interval 0ms, a total of 4000-5000 cycles per day, 1-3 cycles per day, and an action period of 14-21 days.

[0021] A bone tissue regeneration and repair module based on a directly connected electrode cell-carrying biomaterial and an in vitro electrical stimulation sterile culture mold is obtained by using any of the above-mentioned preparation methods.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] 1) The present invention uses PMMA or PET, graphene, MXene, carbon nanotubes and other nano-conductive fillers, sodium alginate, chitosan, PLGA and other adhesives, BMSCs, GelMA, ChiMA, HAMA, AlgMA and other polymer matrices, thermosensitive physical gelatin microspheres and the like as raw materials, and combines blending, coating, 3D printing, in-situ UV curing and other combined processes to provide a method for preparing a bone tissue regeneration and repair module based on directly connected electrode cell-laden biomaterials and an in vitro electrical stimulation sterile culture mold. The above raw material selection and process combination are original to the present invention.

[0024] 2) The in vitro electrical stimulation sterile culture mold in the present invention is independently optimized and designed by the present invention in combination with the defects of the existing electrical stimulation device. The specific structure is: the in vitro electrical stimulation sterile culture mold is provided with two gates at an interval of 180° on the left and right sides, a total of 4 gates, the gate width is 0.5-2cm, the height is 2-3cm, the thickness is 0.5-1mm, and the inner and outer spacing of the two gates on one side is 0.2-0.5cm; the internal cross-sectional dimensions of the embedded groove are consistent with the bottom surface dimensions of the gate, the depth is 400-1000 microns, and the raw materials are all one of PET or PMMA, preferably PET; the cover plate is an outer cover type. The above structure is isolated from the inside and outside through the double gates and the embedded groove structure, and at the same time, the extension of the thin-sheet electrode is guaranteed, which can realize the sterile electrical stimulation culture of the bone tissue regeneration and repair module, and the structure is obviously innovative.

[0025] 3) The direct-connected electrode cell-carrying biomaterial in the present invention is prepared by a combination of processes such as layer-by-layer scraping, blending, 3D printing or in-situ UV curing, and thermosensitive pores, and the material has a sandwich structure. First, nano-conductive fillers such as graphene, MXene, and carbon nanotubes are selected and blended with adhesives such as sodium alginate, chitosan, and PLGA, and then a conductive thin sheet electrode material is formed under a scraping process, which is used to directly and tightly wrap the upper and lower sides of the direct-connected electrode cell-carrying biomaterial; then, thermosensitive physical gelatin microspheres, BMSCs, and polymer matrices such as GelMA, ChiMA, HAMA, and AlgMA are combined for mixing, and 3D printing or in-situ UV curing is combined to form a biomaterial containing thermosensitive physical gelatin microspheres and BMSCs on the bottom conductive thin sheet electrode, and the surface conductive thin sheet electrode is further covered on its surface to form a complete sandwich structure. When the direct-connected electrode cell-carrying biomaterial is cultured at 37°C, the internal gelatin microspheres melt to form a porous structure (i.e., thermosensitive pores). The direct-connected electrode cell-carrying biomaterial prepared by the above-mentioned raw materials and combined processes has thin-film electrodes directly connected to the upper and lower layers. While the middle layer loads the cells, a porous structure is formed for nutrient and material exchange. This form successfully solves the problem of poor electric field application effect caused by the large distance between traditional electrodes and materials. The material preparation technology and structure are innovative.

[0026] 4) The combined strategy of the direct-connected electrode-loaded cell biomaterial and the in vitro electrical stimulation sterile culture mold in the present invention provides a huge boost to the efficiency of electrical stimulation-induced osteogenic differentiation: first, the cell-loaded biomaterial is directly connected to the thin-sheet electrodes on the upper and lower sides, there is no gap between the electrode and the material, and the shape of the electrode and the cell-loaded biomaterial is consistent, and the input electrical stimulation can act on the material completely and evenly, overcoming the problem of too large spacing between traditional electrodes and materials and uneven transmission of electrical stimulation signals; further, the entire direct-connected electrode-loaded cell biomaterial is placed inside the in vitro electrical stimulation sterile culture mold for culture, and only the gate and groove structure allow the thin-sheet electrode to be extended outside the mold. The double gate and groove structure successfully achieves the isolation of the internal and external environments of the mold, as well as the retention of the internal culture medium, providing a highly sterile environment for in vitro differentiation and incubation of the direct-connected electrode-loaded cell biomaterial, and the extended thin-sheet electrode can be connected to components such as a pulsed DC power supply through a wire to form an intelligent electrical stimulation device. The above strategy is designed by the present invention in combination with the requirements of in vitro electrical stimulation to induce directional differentiation of cells.

[0027] 5) The tissue module preparation technology in the present invention is highly efficient, highly safe, highly directional induction capable, highly compatible, customizable, and universal. The shape and size of the bone defect repair material obtained based on CT results and 3D reconstruction can achieve efficient matching with the patient's bone defect area; due to the introduction of the in vitro electrical stimulation sterile culture mold, the high sterile culture environment in the mold and the high safety of the material are guaranteed; at the same time, double conductive thin-film electrodes are introduced on the upper and lower surfaces of the cell-carrying biomaterial and specific pulsed direct current stimulation is applied, which can efficiently and directionally induce the osteogenic differentiation of BMSCs; the above-mentioned material can be applied to various bone defect diseases, with obvious high efficiency and universality. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the preparation process of the bone tissue regeneration and repair module based on the direct-connected electrode cell-carrying biomaterial and the in vitro electrical stimulation sterile culture mold of the present invention;

[0029] Figure 2 In vivo repair Micro-CT results of the bone tissue regeneration and repair module based on the direct-connected electrode-loaded cell biomaterial and the in vitro electrical stimulation sterile culture mold of the embodiments of the present invention (a corresponds to Example 2, b corresponds to Example 1). DETAILED DESCRIPTION

[0030] The present invention is further described below with reference to specific examples and accompanying drawings.

[0031] Comparative Example 1:

[0032] 1) Combine CT scanning to detect the shape of the bone defect area, and perform three-dimensional reconstruction to obtain the size parameters of the initial bone tissue regeneration and repair module. Assuming that the bone defect is a disc with a diameter of 1 cm and a thickness of 1 mm, the shape of the initial bone tissue regeneration and repair module is consistent with the above bone defect shape. Select the commercially available L-shaped platinum electrical stimulation cell culture system for culture.

[0033] 2) The thermosensitive physical gelatin microspheres with a diameter of 50-75 μm (concentration of 0.5 wt%) and BMSCs of the second generation (load concentration of 10 6 -10 7 / ml) of HAMA solution (concentration of 7.5wt%, photoinitiator is LAP, photoinitiator concentration is 0.25wt%). Under sterile conditions, pour the HAMA solution containing cells and gelatin microspheres into the mold of the initial bone tissue regeneration and repair module (inner diameter is 1cm, height is 1mm), and combine with UV light curing process to achieve in situ curing (UV wavelength is 405nm, power is 20W, time is 20s), and the initial bone tissue regeneration and repair module is obtained after demolding. The above-mentioned bone tissue regeneration and repair module is placed in the L-shaped platinum electr small electrical stimulation cell culture system.

[0034] 4) Applying a pulsed direct current electric field (voltage 6V, positive and negative square pulse wave, wave width 1500ms, wave interval 0ms, cycle 4000 times / day, 2 times a day) to obtain a bone tissue regeneration and repair module after 14 days of in vitro culture, which can be used for bone defect regeneration and repair after in situ implantation.

[0035] The bone tissue regeneration and repair model cultured with the above materials and equipment had a survival rate of about 81% combined with live and dead cell fluorescent staining; after multiple cultures, the incidence of contamination was about 40% (i.e., material contamination occurred about four times out of ten cultures); after 14 days, combined with ALP detection, it was found that the ALP activity was about 263u / g protein. It was implanted into a rat skull defect model, and after 28 days of feeding, the sample was taken for Micro-CT, and the results showed that about 72% of the area was covered by new bone.

[0036] Embodiment 1:

[0037] 1) Combine CT scanning to detect the shape of the bone defect area, and perform three-dimensional reconstruction to obtain the size parameters of the initial bone tissue regeneration and repair module and the in vitro electrical stimulation culture sterile mold. Assuming that the bone defect is a disc with a diameter of 1 cm and a thickness of 1 mm, the shape of the initial bone tissue regeneration and repair module is consistent with the above bone defect shape, and the inner diameter of the in vitro electrical stimulation culture sterile mold is about 2 cm and the depth is 2 cm.

[0038] 2) Based on the above parameters, a sterile mold (made of PET) for in vitro electrical stimulation culture was prepared, such as Figure 1 At the same time, two arc-shaped gates that can be pushed and pulled vertically are set on both sides of the mold, with a total of 4 gates (made of PET, the arc gate has an arc length of 0.5cm, a height of 2cm, a thickness of 0.5mm, and an inner and outer distance between the two gates on one side is 0.3cm. The gates on both sides of the mold are on the same straight line). At the same time, the lower end of the gate adopts an embedded groove design (the internal cross-sectional dimensions of the embedded groove are consistent with the bottom surface dimensions of the gate, and the depth is 400 microns); the upper end of the mold is open, and is separated by an outer cover plate.

[0039] 3) Prepare a cell-carrying biomaterial directly connected to a conductive sheet electrode. After mixing 0.25wt% graphene with 2wt% sodium alginate adhesive, a conductive sheet electrode was obtained by a scraping process. Combined with a cutting process, a conductive sheet electrode with a main body consistent with the surface shape of the cell-carrying biomaterial and an electrode sheet with a width of 0.4cm and a length of 1.5cm on both sides was obtained. A total of 2 sheets of conductive sheet electrodes were prepared. The thermosensitive physical gelatin microspheres with a diameter of 50-75 microns (concentration of 0.5wt%) and BMSCs of the second generation (load concentration of 10 6 -10 7 / ml) of HAMA solution (concentration of 7.5wt%, photoinitiator is LAP, photoinitiator concentration is 0.25wt%). Under sterile conditions, take one of the conductive thin sheet electrodes as the bottom layer, and lay a hollow ring-shaped mold, i.e., the mold of the initial bone tissue regeneration and repair module (inner diameter of 1cm, height of 1mm), pour the HAMA solution containing cells and gelatin microspheres into the mold, and lay a second conductive thin sheet electrode on its upper surface, and combine with UV light curing process to achieve in-situ curing (UV wavelength of 405nm, power of 20W, time of 20s), and after demolding, obtain the cell-carrying biomaterial directly connected to the conductive thin sheet electrode, i.e., the initial bone tissue regeneration and repair module. The initial bone tissue regeneration and repair module is placed in an in vitro electrical stimulation culture sterile mold, and the electrode sheet is connected to an external electric field application device from the double gates, so as to realize the connection between the electrode sheet and the electrical stimulation power supply and ensure the internal sterile environment, and together constitute the in vitro electrical stimulation incubation system of the bone tissue regeneration and repair module.

[0040] 4) Applying a bionic pulsed direct current electric field (voltage 6V, positive and negative pulse square wave, wave width 1500ms, wave interval 0ms, a total of 4000 cycles per day, 2 cycles per day) after 14 days of in vitro culture, a bone tissue regeneration and repair module of a directly connected electrode-loaded cell biomaterial and an in vitro electrical stimulation sterile culture mold was obtained, which can be used for bone defect regeneration and repair after in situ implantation.

[0041] Compared with the comparative example, this embodiment uses a self-designed in vitro electrical stimulation culture sterile mold. The conductivity of the conductive sheet electrode is about 5.26S / cm; the bone tissue regeneration repair mold cultured with the above materials and equipment has a survival rate of about 89% combined with live and dead cell fluorescent staining; after multiple cultures, contamination basically does not occur; after 14 days, combined with ALP detection, it is found that the ALP activity is about 389u / g protein, and it is implanted into a rat skull defect model. After 28 days of feeding, the Micro-CT results show that the bone defect is basically completely repaired, such as Figure 2 Middle b.

[0042] Embodiment 2:

[0043] 1) Combine CT scanning to detect the shape of the bone defect area, and perform three-dimensional reconstruction to obtain the size parameters of the initial bone tissue regeneration and repair module and the in vitro electrical stimulation culture sterile mold. Assuming that the bone defect is a disc with a diameter of 1 cm and a thickness of 1 mm, the shape of the initial bone tissue regeneration and repair module is consistent with the above bone defect shape, and the inner diameter of the in vitro electrical stimulation culture sterile mold is about 2 cm and the depth is 2 cm.

[0044] 2) Based on the above parameters, a sterile mold (made of PET) for in vitro electrical stimulation culture was prepared, such as Figure 1 At the same time, two arc-shaped gates that can be pushed and pulled vertically are set on both sides of the mold, with a total of 4 gates (made of PET, the arc gate has an arc length of 0.5cm, a height of 2cm, a thickness of 0.5mm, and an inner and outer distance between the two gates on one side is 0.3cm. The gates on both sides of the mold are on the same straight line). At the same time, the lower end of the gate adopts an embedded groove design (the internal cross-sectional dimensions of the embedded groove are consistent with the bottom surface dimensions of the gate, and the depth is 400 microns); the upper end of the mold is open, and is separated by an outer cover plate.

[0045] 3) Prepare a cell-carrying biomaterial directly connected to a conductive sheet electrode. After mixing 0.25wt% of graphene and 2wt% of sodium alginate binder, a conductive sheet electrode was obtained by a doctor blade process. Combined with a cutting process, a conductive sheet electrode with a main body consistent with the surface shape of the cell-carrying biomaterial and an electrode sheet with a width of 0.4cm and a length of 1.5cm on both sides was obtained. A total of 2 sheets of conductive sheet electrodes were prepared. 6 -10 7 / ml) of HAMA solution (concentration of 7.5wt%, photoinitiator is LAP, photoinitiator concentration is 0.25wt%). Under sterile conditions, take one of the conductive thin sheet electrodes as the bottom layer, and lay a hollow ring-type mold, i.e., the mold of the initial bone tissue regeneration and repair module (inner diameter of 1cm, height of 1mm), pour the cell-carrying HAMA solution into the mold, and lay a second conductive thin sheet electrode on its upper surface, and combine with UV light curing process to achieve in-situ curing (UV wavelength of 405nm, power of 20W, time of 20s), and after demolding, obtain the cell-carrying biomaterial directly connected to the conductive thin sheet electrode, i.e., the initial bone tissue regeneration and repair module. The initial bone tissue regeneration and repair module is placed in an in vitro electrical stimulation culture sterile mold, and the electrode sheet is connected to an external electric field application device from the double gates, so as to realize the connection between the electrode sheet and the electrical stimulation power supply and ensure the internal sterile environment, together forming an in vitro electrical stimulation incubation system for the bone tissue regeneration and repair module.

[0046] 4) Applying a bionic pulsed direct current electric field (voltage 6V, positive and negative pulse square wave, wave width 1500ms, wave interval 0ms, a total of 4000 cycles per day, 2 cycles per day) to obtain a bone tissue regeneration and repair module after 14 days of in vitro culture, which can be used for bone defect regeneration and repair after in situ implantation.

[0047] Compared with Example 1, this example does not add physical gelatin microspheres in the bone tissue regeneration and repair module. The conductivity of the conductive sheet electrode is about 5.31S / cm; the bone tissue regeneration and repair model cultured with the above materials and equipment has a survival rate of about 58% combined with live and dead cell fluorescent staining; after multiple cultures, contamination basically does not occur; after 14 days, combined with ALP detection, it is found that the ALP activity is about 174u / g protein, and it is implanted into a rat skull defect model. After 28 days of feeding, the Micro-CT results show that about 48% of the area is covered by new bone, such as Figure 2 Middle a.

[0048] Embodiment 3:

[0049] 1) Combine CT scanning to detect the shape of the bone defect area, and perform three-dimensional reconstruction to obtain the size parameters of the initial bone tissue regeneration and repair module and the in vitro electrical stimulation culture sterile mold. Assuming that the bone defect is a disc with a diameter of 1 cm and a thickness of 1 mm, the shape of the initial bone tissue regeneration and repair module is consistent with the above bone defect shape, and the inner diameter of the in vitro electrical stimulation culture sterile mold is about 2 cm and the depth is 2 cm.

[0050] 2) Based on the above parameters, an in vitro electrical stimulation culture sterile mold (made of PET) was prepared, and a vertically push-pull arc gate was set on both sides of the mold, with a total of 2 gates (made of PET, the arc gate has an arc length of 0.5 cm, a height of 2 cm, and a thickness of 0.5 mm. The gates on both sides of the mold are on the same straight line). At the same time, the lower end of the gate adopts an embedded groove design (the internal cross-sectional dimensions of the embedded groove are consistent with the bottom surface dimensions of the gate, and the depth is 400 microns); the upper end of the mold is open, and an outer cover plate is provided to separate it.

[0051] 3) Prepare a cell-carrying biomaterial directly connected to a conductive sheet electrode. After mixing 0.25wt% graphene with 2wt% sodium alginate adhesive, a conductive sheet electrode was obtained by a scraping process. Combined with a cutting process, a conductive sheet electrode with a main body consistent with the surface shape of the cell-carrying biomaterial and an electrode sheet with a width of 0.4cm and a length of 1.5cm on both sides was obtained. A total of 2 sheets of conductive sheet electrodes were prepared. The thermosensitive physical gelatin microspheres with a diameter of 50-75 microns (concentration of 0.5wt%) and BMSCs of the second generation (load concentration of 10 6 -10 7 / ml) of HAMA solution (concentration of 7.5wt%, photoinitiator is LAP, photoinitiator concentration is 0.25wt%). Under sterile conditions, take one of the conductive thin sheet electrodes as the bottom layer, and lay a hollow ring-type mold, i.e., the mold of the initial bone tissue regeneration and repair module (inner diameter of 1cm, height of 1mm), pour the HAMA solution containing cells and gelatin microspheres into the mold, and lay a second conductive thin sheet electrode on its upper surface, and combine with UV light curing process to achieve in situ curing (UV wavelength of 405nm, power of 20W, time of 20s), and after demolding, obtain the cell-carrying biomaterial directly connected to the conductive thin sheet electrode, i.e., the initial bone tissue regeneration and repair module. The initial bone tissue regeneration and repair module is placed in an in vitro electrical stimulation culture sterile mold, and the electrode sheet is connected to an external electric field application device from the gate, so as to realize the connection between the electrode sheet and the electrical stimulation power supply and ensure the internal sterile environment, together forming an in vitro electrical stimulation incubation system for the bone tissue regeneration and repair module.

[0052] 4) Applying a bionic pulsed direct current electric field (voltage 6V, positive and negative pulse square wave, wave width 1500ms, wave interval 0ms, a total of 4000 cycles per day, 2 cycles per day) to obtain a bone tissue regeneration and repair module after 14 days of in vitro culture, which can be used for bone defect regeneration and repair after in situ implantation.

[0053] Compared with Example 1, the number of gates in the in vitro electrical stimulation culture sterile mold is reduced in this embodiment, and the number of gates on both sides is reduced to one. The conductivity of the conductive sheet electrode is about 5.10S / cm; the survival rate of the bone tissue regeneration repair mold cultured with the above materials and equipment combined with the live and dead cell fluorescent staining cells is about 86%; after multiple cultures, the incidence of contamination is about 10%; after 14 days, combined with ALP detection, it is found that the ALP activity is about 361u / g protein, and it is implanted into a rat skull defect model. After 28 days of feeding, the results of Micro-CT taken out of the sample show that the bone defect is basically completely repaired.

Claims

1. A method for preparing a bone tissue regeneration and repair module based on a directly connected electrode-loaded cell biomaterial and an in vitro electrical stimulation sterile culture mold, characterized in that: The following steps are involved: 1) obtaining the size parameters of the initial bone tissue regeneration and repair module and the in vitro electrical stimulation culture sterile mold as needed, and casting a mold of the initial bone tissue regeneration and repair module; 2) preparing the in vitro electrical stimulation culture sterile mold based on the obtained size parameters, wherein the in vitro electrical stimulation culture sterile mold is a hollow cylinder composed of two concentric circles of different diameters, and arc gates capable of vertical push-pull switches are respectively arranged on both sides of each concentric circle of the mold, and the lower end of the gate adopts an embedded groove design; the top of the mold is open and is provided with a cover plate for separation; 3) Prepare a cell-carrying biomaterial directly connected to a conductive sheet electrode, and connect an electric field application device to the double arc gates through the conductive sheet electrode to form an in vitro electrical stimulation incubation system for bone tissue regeneration and repair module. ; 4) After 14-21 days of in vitro culture by applying a bionic pulsed direct current electric field, a bone tissue regeneration and repair module is obtained.

2. The method for preparing a bone tissue regeneration and repair module based on a directly connected electrode-loaded cell-bearing biomaterial and an in vitro electrical stimulation sterile culture mold according to claim 1, characterized in that: In step 1), the inner diameter of the sterile mold for in vitro electrical stimulation culture is 1-3 cm larger than the maximum linear dimension of the bone tissue regeneration and repair module.

3. The method for preparing a bone tissue regeneration and repair module based on a directly connected electrode-loaded cell-bearing biomaterial and an in vitro electrical stimulation sterile culture mold according to claim 1, characterized in that: In step 2), the arc length of the arc gate is 0.5-2 cm, the height is 2-3 cm, the thickness is 0.5-1 mm, the distance between the two gates on the same side is 0.2-0.5 cm, and the connection line of the four gates is the same straight line; The internal cross-sectional dimensions of the embedded groove are consistent with the bottom dimensions of the gate, with a depth of 400-1000 microns, and the raw materials are either PET or PMMA; The cover plate is an outer cover type.

4. The method for preparing a bone tissue regeneration and repair module based on a directly connected electrode-loaded cell-bearing biomaterial and an in vitro electrical stimulation sterile culture mold according to claim 1, characterized in that: In step 3), during the preparation of the cell-carrying biomaterial directly connected to the conductive sheet electrode, the cell-carrying biomaterial is placed on the conductive sheet electrode, and another conductive sheet electrode is laid on the upper surface thereof to obtain the cell-carrying biomaterial directly connected to the conductive sheet electrode.

5. The method for preparing a bone tissue regeneration and repair module based on a directly connected electrode cell-carrying biomaterial and an in vitro electrical stimulation sterile culture mold according to claim 1, characterized in that: In step 3), the conductive sheet electrode is prepared by blending a nano conductive filler and a binder and then using a doctor blade method; the nano conductive filler is one of graphene, MXene, and carbon nanotubes, with a concentration of 0.05-0.25wt%; the binder is one or more of sodium alginate, chitosan, and PLGA, with a concentration of 1-5wt%; the conductive sheet electrode has a thickness of 200-600 microns, a shape consistent with that of the cell-carrying biomaterial, and each sheet has an electrode sheet extension with a width of 0.4-1.8 cm and a length of 0.5-2 cm.

6. The method for preparing a bone tissue regeneration and repair module based on a directly connected electrode cell-carrying biomaterial and an in vitro electrical stimulation sterile culture mold according to claim 5, characterized in that: In step 3), during the preparation of the cell-loaded biomaterial, in situ UV light curing or 3D printing is used to prepare a solution containing cells and gelatin microspheres, wherein the solution contains a photoinitiator, the cells are bone marrow mesenchymal stem cells (BMSCs), the number of generations is 2-5, and the loading concentration is 10 6 -10 7 / ml; the carrier of the solution is one or more of methacryloyl gelatin (GelMA), methacryloyl chitosan (ChiMA), methacryloyl hyaluronic acid (HAMA), and methacryloyl sodium alginate (AlgMA), with a concentration of 5-15wt%; the photoinitiator is one or more of LAP, Irgacure, and photoinitiator 2959, with a concentration of 0.1-0.4wt%; the gelatin microspheres are temperature-sensitive physical gelatin microspheres with a diameter of 50-200 microns and a concentration of 0.5-1.5wt%, which are used to form a porous structure of a cell-carrying biomaterial.

7. The method for preparing a bone tissue regeneration and repair module based on a directly connected electrode cell-carrying biomaterial and an in vitro electrical stimulation sterile culture mold according to claim 6, characterized in that: During the in-situ UV light curing process, the solution containing cells and gelatin microspheres is poured into the mold of the initial bone tissue regeneration and repair module, with a UV wavelength of 405 nm, a power of 10-20 W, and a time of at least 20 seconds.

8. The method for preparing a bone tissue regeneration and repair module based on a directly connected electrode cell-carrying biomaterial and an in vitro electrical stimulation sterile culture mold according to claim 1, characterized in that: In step 4), the bionic pulsed DC electric field action parameters applied include: voltage 3-8V, positive and negative pulse square wave, wave width 1500-2500ms, wave interval 0ms, a total of 4000-5000 cycles per day, 1-3 cycles per day, and an action period of 14-21 days.

9. A bone tissue regeneration and repair module based on directly connected electrode-loaded cell biomaterials and an in vitro electrical stimulation sterile culture mold, characterized in that: The method is adopted as described in any one of claims 1 to 8.

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