In-vitro three-dimensional cell electrical stimulation osteogenesis induction system based on functionalized GelMA hydrogel

Abstract

The invention discloses an in-vitro three-dimensional cell electrical stimulation osteogenesis induction system based on functionalized GelMA hydrogel. The in-vitro three-dimensional cell electrical stimulation osteogenesis induction system comprises a hydrogel three-dimensional culture platform and an electrical stimulation device, wherein the hydrogel three-dimensional culture platform is constructed by wrapping cells with composite conductive hydrogel, and the electrical stimulation device is used for providing an electrical stimulation function for the hydrogel three-dimensional culture platform. The system is composed of the 3D culture platform based on the composite conductive hydrogel and the electrical stimulation device composed of the cell culture pore plate, the 3D culture platform and the electrical stimulation device form an in-vitro 3D electrical microenvironment system, and compared with a traditional 2D culture system, the system can further simulate an in-vivo endogenous environment of cells, so that the in-vivo endogenous environment of the cells is simulated, and the cell culture efficiency is improved. And osteogenic differentiation, proliferation and the like of the cells are promoted through electrical stimulation, so that more simulated mechanism research on osteogenic differentiation promoted by electrical signals can be realized, and an integrated platform from cell culture to final cell use is provided.

Description

Technical Field

[0001] The invention relates to the field of bioengineering, and in particular to an in vitro three-dimensional cell electrical stimulation osteogenic induction system based on functionalized GelMA hydrogel. Background Art

[0002] Stem cells are the most common type of cells in tissue repair and regeneration. They have the ability to self-renew, multidirectionally differentiate, and regulate the immune system. They can be directly isolated from a variety of mature organs, such as bone marrow, adipose tissue, and blood, and are a type of cell with great application prospects in tissue engineering. Among them, bone marrow-derived mesenchymal stem cells can differentiate into a variety of tissue cells under specific induction conditions in vivo or in vitro. They have a wide range of sources, are easy to separate and culture, and have strong differentiation potential and can be autologously transplanted. They are an ideal cell source for regenerative medicine, especially for articular cartilage repair.

[0003] The cell microenvironment is closely related to the fate of mesenchymal stem cells (MSCs). Due to its natural piezoelectric properties, bone tissue exposes MSCs to an endogenous electrical microenvironment, which has been shown to be essential for cell growth, survival, differentiation, and morphological changes. However, most studies on the effects of electrical stimulation on MSC differentiation are conducted on the surface of 2D materials. Two-dimensional culture conditions cannot truly reproduce the three-dimensional structure of the natural microenvironment. Cells lose the interaction between cells and cells and cells and ECM in the in vivo environment under 2D culture conditions, and show different functional and morphological characteristics. Drug screening based on 2D cultured cells often encounters inconsistencies in in vivo and in vitro efficacy, which changes their behavior under two-dimensional culture conditions and cannot well mimic the natural structure of the BMSC microenvironment. The 3D culture environment is a condition closer to the body. 2D culture allows cells to only receive physical signals from the matrix from the back, while 3D cultured cells can receive physical signals from the matrix from 360°. The purpose of the 3D cell culture system is to simulate the in situ function of living tissues by reconstructing tissue structure and providing a favorable microenvironment for cell survival, proliferation, and differentiation, which is more similar to the in vivo environment. Therefore, constructing an in vitro 3D electrical microenvironment biomimetic platform can be closer to and simulate the in vivo conditions of bone marrow mesenchymal stem cells, provide a more realistic platform for in vitro research on the interaction and mechanism between cells and matrix, and optimize the integrated process from cell culture to final use. However, there is no publicly available reliable solution. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an in vitro three-dimensional cell electrical stimulation osteogenic induction system based on functionalized GelMA hydrogel in view of the deficiencies in the above-mentioned prior art. The present invention uses a composite conductive hydrogel with excellent biocompatibility and stability to build an in vitro 3D cell culture platform, and combines it with an external electrical stimulation device to construct an in vitro 3D culture system that simulates the in vivo 3D electrical microenvironment, providing a more simulated platform for in vitro research on the interaction between cells and matrices and their related mechanisms, especially cell osteogenic induction.

[0005] To achieve the above-mentioned purpose, the technical solution adopted by the present invention is: an in vitro three-dimensional cell electrical stimulation osteogenic induction system based on functionalized GelMA hydrogel, comprising: a hydrogel three-dimensional culture platform formed by wrapping cells with a composite conductive hydrogel, and an electrical stimulation device for providing electrical stimulation function to the hydrogel three-dimensional culture platform;

[0006] The composite conductive hydrogel of the present invention comprises: methacrylate modified gelatin (GelMA), conductive polymer polypyrrole nanowires (ppy nanowires, also known as ppyNWs) constituting a conductive network, and a high molecular polymer (PEGDA) for enhancing the stability of the polymer platform.

[0007] The hydrogel three-dimensional culture platform is constructed by the following method:

[0008] S1. Preparation of GelMA;

[0009] S2, preparing ppy nanowires;

[0010] S3, preparing PEGDA;

[0011] S4. Preparation of hydrogel precursor solution:

[0012] S4-1. Construction of hydrogel three-dimensional culture platform:

[0013] S4-2, dissolving GelMA in a photoinitiator solution, then adding PEGDA, stirring until completely dissolved, then adding ppy nanowires, and ultrasonically dispersing to obtain a hydrogel precursor solution;

[0014] S4-3. Adding cells for culture into a solution of a hydrogel precursor and irradiating the solution with ultraviolet light to solidify the hydrogel precursor into a composite conductive hydrogel and encapsulate the cells, thereby constructing the hydrogel three-dimensional culture platform.

[0015] Preferably, step S1 specifically comprises:

[0016] 10 g of type A gelatin solid powder was added to 100 mL of PBS solution, stirred at 60 °C for 2 h, 6 mL of methacrylic anhydride was added dropwise at a rate of 0.5 mL / min under light protection, and the reaction was allowed to proceed for 3 h. PBS solution preheated to 37 °C was added to terminate the reaction. The product was dialyzed in deionized water for 7 days using an 8K-14K Da dialysis bag, and the solution in the dialysis bag was freeze-dried to obtain GelMA.

[0017] Preferably, step S2 specifically comprises:

[0018] Take 0.1g of hexadecyltrimethylammonium bromide and add it to 30mL of 0.5M hydrochloric acid solution. After stirring until it is completely dissolved, 0.3mL of pyrrole monomer is added dropwise at a rate of 0.4mL / min. Stir evenly, add 30mL of 0.5M HCL solution containing 0.2g of ammonium persulfate, react at 4°C and 500rpm for 8 hours, centrifuge at 11000rpm, collect the solid product, rinse with deionized water and anhydrous ethanol in turn, and dry to obtain ppy nanowires.

[0019] Preferably, step S3 specifically comprises:

[0020] S3-1, take 10-40g of PEG solid with a molecular weight of 2000 and add it to 30-120mL of dichloromethane, stir until completely dissolved, add 0.2-0.8mL of triethylamine and 0.35-1.4mL of acryloyl chloride in sequence at 0-10°C, and then react under a nitrogen atmosphere for 24-96 hours;

[0021] S3-2. After the reaction is completed, 10-40 mL of 1-4 mol / L potassium carbonate solution is added to the product, and the resulting reaction solution is transferred to a separatory funnel. After shaking, the reaction solution is allowed to stand for stratification. The dichloromethane phase reaction solution at the bottom is taken, and magnesium sulfate powder is added to remove water. The supernatant is collected by centrifugation and concentrated by rotary evaporation. Diethyl ether is added to the resulting concentrated solution until a white powder solid is precipitated. The concentrated solution is filtered and the collected white solid sample is vacuum dried at 20-40°C to constant weight to obtain a crude PEGDA product.

[0022] S3-3. The crude PEGDA product was dialyzed in deionized water for 36-144 hours using a dialysis bag and freeze-dried to obtain purified PEGDA.

[0023] Preferably, step S3 specifically comprises:

[0024] S3-1, take 20g of PEG solid with a molecular weight of 2000 and add it to 60mL of dichloromethane, stir until completely dissolved, add 0.4mL of triethylamine and 0.7mL of acryloyl chloride in sequence at 4°C, and then react for 48 hours under a nitrogen atmosphere;

[0025] S3-2. After the reaction is completed, 20 mL of 2 mol / L potassium carbonate solution is added to the product, and the resulting reaction solution is transferred to a separatory funnel. After shaking, the reaction solution is allowed to stand for stratification. The dichloromethane phase reaction solution at the bottom is taken, and magnesium sulfate powder is added several times to remove water. The supernatant is collected by centrifugation and concentrated by rotary evaporation. Ether is added to the resulting concentrated solution until a white powder solid is precipitated. The collected white solid sample is transferred to a 25°C vacuum drying oven and dried to constant weight to obtain a crude PEGDA product.

[0026] S3-3. The crude PEGDA product was dialyzed in deionized water for 72 hours using a dialysis bag with a molecular weight cutoff of 2000 Da, and freeze-dried at -20°C to obtain purified PEGDA.

[0027] Preferably, the photoinitiator solution is prepared by preparing a photoinitiator LAP and a PBS solution.

[0028] Preferably, step S4 is specifically:

[0029] S4-2, add 50-200 mg of photoinitiator LAP into 50-200 mL of PBS solution, stir at 40-60° C. in dark for 0.5-2 hours to obtain a photoinitiator solution;

[0030] Take 0.1875-0.75g of GelMA prepared in step S1 and add it to 1-5mL of photoinitiator solution, stir at 40-60°C until it is completely dissolved, then add 1wt%-2wt% of PEGDA prepared in step S3, stir for 1.5-6h, then add 0.5wt%-1.5wt% of ppy nanowires prepared in step S2, stir and react for 1.5-6h to obtain a hydrogel precursor solution;

[0031] S4-3, adding cells for culture into the solution of hydrogel precursor, irradiating with 405nm ultraviolet light for 45-180s, so that the hydrogel precursor is solidified into a composite conductive hydrogel and wraps the cells, thereby constructing the hydrogel three-dimensional culture platform.

[0032] Preferably, step S4 is specifically

[0033] S4-2, add 100 mg of photoinitiator LAP into 100 mL of PBS solution, stir at 50° C. in dark for 1 hour to obtain a photoinitiator solution;

[0034] 0.375 g of GelMA prepared in step S1 was added to 2.5 mL of photoinitiator solution, and stirred at 50° C. until completely dissolved. Then, 1 wt% to 2 wt% of PEGDA prepared in step S3 was added, and after stirring for 3 h, 0.5 wt% to 1.5 wt% of ppy nanowires prepared in step S2 were added, and the mixture was stirred for reaction for 3 h to obtain a hydrogel precursor solution.

[0035] S4-3. Add the cells to be cultured into the solution of the hydrogel precursor, and irradiate with 405nm ultraviolet light for 90s to solidify the hydrogel precursor into a composite conductive hydrogel and wrap the cells, thereby constructing the hydrogel three-dimensional culture platform.

[0036] Preferably, the electrical stimulation device comprises a cell culture well plate, a positive electrode and a negative electrode arranged in the culture wells of the cell culture well plate, and an electrical stimulation host connected to the positive electrode and the negative electrode for providing an electrical stimulation function.

[0037] More preferably, the cell culture plate is a 12-well cell culture plate, and each independent culture well is provided with two through holes at the top, which are positive and negative electrode holes, respectively, for inserting positive and negative electrodes (both are 0.5mm pure platinum wire electrodes), and the electrodes of each well are connected in series, and then connected to the electrical stimulation host, and electrical signal stimulation is performed according to demand to form a 3D electrical microenvironment culture system. The positive electrode and the negative electrode are vertically inserted into the composite conductive hydrogel (i.e., the hydrogel three-dimensional culture platform) wrapped with cells, and the hydrogel three-dimensional culture platform in each different group of culture wells simulates different microenvironments in the body, forming an experimental control, and obtaining different experimental results.

[0038] Preferably, the electrical stimulation method of the in vitro three-dimensional cell electrical stimulation osteogenic induction system based on functionalized GelMA hydrogel is:

[0039] The hydrogel three-dimensional culture platform prepared in step S4 is transferred to the culture well of the cell culture well plate. After the culture well is washed once with culture medium, fresh complete culture medium is added to the culture well, and the well is placed in a 37°C incubator containing 5% CO2 for static culture. The culture medium is replaced every 2-3 days. During the culture process, the electric stimulation host applies electric stimulation to the hydrogel three-dimensional culture platform in the culture well through the positive electrode and the negative electrode. The voltage of the electric stimulation is 1-5V, and the time of the electric stimulation is 20-80min per day.

[0040] The beneficial effects of the present invention are:

[0041] The present invention provides an in vitro three-dimensional cell electrical stimulation osteogenic induction system based on functionalized GelMA hydrogel. The system of the present invention is composed of a 3D culture platform based on a composite conductive hydrogel and an electrical stimulation device composed of a cell culture well plate. The two constitute an in vitro 3D electrical microenvironment system. Compared with the traditional 2D culture system, the system can further simulate the endogenous environment of the cell in vivo, and promote the osteogenic differentiation and proliferation of the cell through electrical stimulation, so as to realize more simulated research on the mechanism of electrical signal promoting osteodifferentiation, and provide an integrated platform from cell culture to final cell use. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The preparation process of the system of the present invention: (A) the synthesis route of GelMA, (B) the synthesis route of ppyNWs, (C) the physical picture of the electric stimulation device, (D) the schematic diagram of the construction of the electric stimulation microenvironment system;

[0043] Figure 2 The performance test results of the conductive hydrogel in the present invention are: (A) scanning electron microscopy image of ppyNWs; (B) infrared image and of GelMA; (C) nuclear magnetic hydrogen spectrum of GelMA; (D) compression modulus of hydrogels with different PEGDA contents; (E) stress-strain curves and of different conductive hydrogels; (F) compression modulus of different conductive hydrogels; (G) scanning electron microscopy image of conductive hydrogel; (H) conductivity of conductive hydrogel; (I) swelling properties of hydrogel; (J) degradation properties of hydrogel.

[0044] Figure 3 The test results of the influence of the system of the present invention on cell compatibility are as follows: (A) Fluorescence image of FDA / PI staining (green-live cells, red-dead cells), scale bar 200 μm; (B) relative cell viability;

[0045] Figure 4 Results of cell proliferation tests using the system of the present invention: whether cells were subjected to electrical stimulation in the hydrogel and the selection of the optimal electrical stimulation voltage (A), the selection of the optimal electrical stimulation time (B), and the cell proliferation rate after seven days of stimulation under the optimal electrical stimulation parameters (C).

[0046] Figure 5 The test results of the system of the present invention on the osteogenic differentiation of cells: alkaline phosphatase staining and activity analysis of cells in hydrogels with and without electrical stimulation for 4 and 7 days (A, D); collagen staining and quantitative analysis for 7 and 14 days (B, E); immunofluorescence staining and quantitative analysis for 7 days (C, F). DETAILED DESCRIPTION

[0047] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0048] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0049] The test methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified. In the following examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0050] Embodiment 1

[0051] An in vitro three-dimensional cell electrical stimulation osteogenic induction system based on functionalized GelMA hydrogel, comprising: a hydrogel three-dimensional culture platform formed by wrapping cells with a composite conductive hydrogel, and an electrical stimulation device for providing electrical stimulation function to the hydrogel three-dimensional culture platform;

[0052] 1. The hydrogel three-dimensional culture platform was constructed by the following method:

[0053] S1. Preparation of GelMA (methacrylate modified gelatin):

[0054] 10 g of type A gelatin solid powder (purchased from Aladdin, brand 9000-70-8) was weighed on a precision balance and transferred to a 200 ml round-bottom flask, added to 100 ml PBS solution, stirred at 60 °C for 2 h to fully dissolve it, 6 ml of methacrylic anhydride (MA) was slowly added dropwise at a rate of 0.5 ml / min under light protection, reacted for 3 h, and 2 volumes (200 ml) of PBS solution preheated to 37 °C were added to terminate the reaction. The product was dialyzed in deionized water for 7 days using a 10K Da dialysis bag to remove unreacted MA monomers, and the solution in the dialysis bag was transferred to multiple 50 ml centrifuge tubes, placed at -80 °C for 48 h, and then freeze-dried in a freeze dryer for 7 days to obtain a white foamy substance: GelMA, which was stored at -20 °C for subsequent experiments.

[0055] S2. Preparation of ppy nanowires (i.e., ppyNWs, conductive polymer polypyrrole nanowires) by soft template method:

[0056] Take 0.1g of hexadecyltrimethylammonium bromide (CTAB) and add it to 30mL of 0.5M hydrochloric acid solution. After stirring until completely dissolved, 0.3mL of pyrrole monomer ((purchased from McLean Company, brand number 109-97-7)) is slowly added dropwise at a rate of 0.4mL / min, and stirred evenly. 30mL of 0.5M HCL solution containing 0.2g of ammonium persulfate (APS) is added to initiate the polymerization of pyrrole monomer. The reaction is carried out at 4°C and 500rpm for 8 hours. The solid product is centrifuged at 11000rpm, and the solid product is collected. It is rinsed with deionized water and anhydrous ethanol three times in sequence to remove impurities and dried to constant weight to obtain ppy nanowires.

[0057] S3. Preparation of PEGDA (polymer):

[0058] S3-1, take 20g of PEG solid with a molecular weight of 2000 into a 250ml round-bottom flask, add 60mL of dichloromethane (dehydrated), stir until completely dissolved, add 0.4mL of triethylamine (dehydrated) and 0.7mL of acryloyl chloride in sequence at 4°C, and then react under a nitrogen atmosphere for 48 hours;

[0059] S3-2. After the reaction is completed, 20 mL of 2 mol / L potassium carbonate solution is added to the product to remove HCL. The resulting reaction solution is transferred to a separatory funnel, which is fully shaken and allowed to stand for stratification. The dichloromethane phase reaction solution at the bottom is taken, and magnesium sulfate powder is added several times to remove water. The supernatant is collected by centrifugation and concentrated by rotary evaporation. Ether is added to the resulting concentrated solution until a white powder solid is precipitated. The concentrated solution is filtered and the collected white solid sample is transferred to a 25°C vacuum drying oven and dried to constant weight to obtain a crude PEGDA product.

[0060] S3-3, the crude PEGDA product was dialyzed in deionized water for 72 hours using a dialysis bag with a molecular weight cutoff of 2000Da, and freeze-dried at -20°C to obtain purified white solid PEGDA; the reaction by-products were completely removed by purification for subsequent cell culture experiments;

[0061] S4. Preparation of hydrogel precursor solution:

[0062] S4-2, weigh 100 mg of the photoinitiator LAP with a precision balance and add it to a 250 ml round-bottom flask, then add it to 100 mL of PBS solution, stir at 50°C in the dark for 1 hour to completely dissolve it in the solution, and prepare a photoinitiator solution with a concentration of 0.1% (w / v), and store it at 4°C in the dark;

[0063] 0.375 g of the foamy white GelMA prepared in step S1 was added to 2.5 mL of the photoinitiator solution, and stirred at 50° C. until completely dissolved. Then, different concentrations (1 wt %, 2 wt %) of PEGDA prepared in step S3 were added. After stirring for 3 h, different concentrations (0, 0.5 wt %, 1 wt %, 1.5 wt %) of ppy nanowires prepared in step S2 were added. The mixture was stirred for 3 h to obtain several groups of hydrogel precursor solutions with different PEGDA and ppy nanowire contents.

[0064] S4-3. Add the cells to be cultured into the solution of the hydrogel precursor and irradiate with 405nm ultraviolet light for 90s to solidify the hydrogel precursor into a composite conductive hydrogel and encapsulate the cells, thereby constructing several groups of hydrogel three-dimensional culture platforms with different PEGDA and ppy nanowire contents for subsequent experiments.

[0065] 2. Electrical stimulation device

[0066] Drill two holes on the top of the 12-well cell culture plate, which are the positive and negative electrode holes, respectively. The spacing between the holes is 1.5 cm to ensure that the intensity of the electrical stimulation applied to each hole is the same. 0.5 mm diameter pure platinum wire electrodes are inserted into the positive and negative electrodes respectively as the positive and negative electrodes. The height of the electrodes exposed above the hole cover is 5 mm. The electrodes are connected in series through copper wires and then connected to the electrical stimulation host. In this embodiment, an electrochemical workstation is used as the electrical stimulation host to apply electrical stimulation, which can easily adjust the electrical stimulation parameters.

[0067] The electrical stimulation method of this system is:

[0068] The hydrogel three-dimensional culture platform prepared in step S4 is transferred to the culture well of the cell culture well plate. After the culture well is washed once with culture medium, fresh complete culture medium is added to the culture well, and the well is placed in a 37°C incubator containing 5% CO2 for static culture. The culture medium is replaced every 2 days. During the culture process, the electric stimulation host applies electric stimulation to the hydrogel three-dimensional culture platform in the culture well through the positive electrode and the negative electrode.

[0069] 3. Performance Testing and Characterization

[0070] 1. Structural characterization: The freeze-dried GelMA sample was quenched with liquid nitrogen, the surface was gold-sprayed, and SEM characterization was performed.

[0071] 2. H-NMR characterization: Add the freeze-dried GelMA sample into a clean NMR tube, add 0.6 ml of deuterated water (D2O), cover it and shake it gently to fully dissolve it, perform H-NMR detection, and analyze the results using MestReNova software.

[0072] 3. Mechanical properties test: A cylindrical hydrogel with a diameter of 4 mm and a thickness of 5 mm was synthesized using an Instalon 5567 (MA, USA) platform in a compression test mode. The deformation rate was 1 mm / min, the compression strain rate was 60%, and the stress-strain curve was obtained. The slope of the stress-strain curve at 5%-10% was used to obtain the compression modulus.

[0073] 4. Electrical property test: The conductivity of the hydrogel was measured by a digital multimeter. To this end, each group of hydrogel sheets with a diameter of 8 mm and a thickness of 1 mm were prepared, copper foil conductive adhesive was attached to two glass plates, and then the hydrogel was placed between them, and its resistance was measured by a digital multimeter. σ represents conductivity, I represents hydrogel thickness, S represents area, and R represents resistance measured by the multimeter.

[0074] 5. Swelling performance: After synthesis, each group of hydrogels was freeze-dried and weighed as initial weight Wd, transferred to PBS solution and soaked for 48 hours, weighed as Wi, and the swelling rate of the hydrogel was calculated by the formula: swelling rate = (Wi-Wd) / Wd×100%.

[0075] In vitro degradability: The synthesized hydrogels of each group were weighed as the initial weight, and then immersed in PBS solution and placed in a 37°C constant temperature shaker. The hydrogels were taken out at each time point to absorb the surface moisture and weigh them.

[0076] Test and Characterization Results

[0077] (1) The scanning electron micrograph of polypyrrole (2A) shows that it has a linear structure with a diameter of about 50 nm and can form a permeable network in the hydrogel network;

[0078] Figure 2 B and C are the infrared and nuclear magnetic proton spectra of GelMA, respectively, which show that GelMA has a peak at 3294 cm -1 、1629cm -1 and 1538cm -1 The characteristic peaks and new absorption peaks at 5.5ppm and 5.7ppm indicate its successful preparation;

[0079] Depend on Figure 2 D obtained that the addition of only 2% PEGDA can increase the compression modulus of the hydrogel to 14 kPa, ensuring that the stability and mechanical strength requirements of the hydrogel for in vitro culture matrix can be met to a certain extent while adding as little PEGDA as possible, while ensuring that the matrix can be degraded and cells can be collected in the presence of collagenase in later experiments.

[0080] Figure 2 In D, 15% GelMA represents the hydrogel without adding PEGDA and ppy nanowires, 15% GelMA / 1% PEGDA and 15% GelMA / 2% PEGDA represent the hydrogel with adding PEGDA with a mass concentration of 1% and 2%, respectively;

[0081] Figure 2 E represents the stress-strain curves of different conductive hydrogels. In the figure, 15% GelMA represents the hydrogel without adding PEGDA and ppy nanowires, 15% GelMA / PEGDA represents the hydrogel with adding PEGDA with a mass concentration of 2% but without adding ppy nanowires, 15% GelMA / PEGDA / 1ppy, 15% GelMA / PEGDA / 1.5ppy, 15% GelMA / PEGDA / 2ppy represent the hydrogel with adding PEGDA with a mass concentration of 2% and 1% of ppy nanowires, the hydrogel with adding PEGDA with a mass concentration of 2% and 1.5% of ppy nanowires, and the hydrogel with adding PEGDA with a mass concentration of 2% and 2% of ppy nanowires, respectively;

[0082] With the addition of polypyrrole, the mechanical properties of the conductive hydrogel are further improved ( Figure 2 F); From the cross-sectional scan of the hydrogel, it can be seen that it forms a uniform pore size, and a conductive network interspersed between them can be observed ( Figure 2 G); conductivity as Figure 2 As shown in Figure 1, the conductivity of GelMA hydrogel is low, 0.052 ms / cm. As the content of ppy nanowires increases, the conductivity of the hydrogel increases significantly, increasing to 0.092 ms / m. This result shows that the addition of ppy nanowires can effectively enhance the conductivity of the hydrogel.

[0083] The swelling ratio of each group of hydrogels within 48h ( Figure 2 I), which rapidly swelled within 10 hours and reached swelling equilibrium in 20 hours. The swelling rate of each group of hydrogels was maintained in the range of 550%-650%, indicating that the hydrogel has an effective material exchange capacity. During the 14-day degradation period, the degradation rate of the hydrogel was relatively stable, and the degradation of the hydrogel was more significant within the first 10 days. Compared with pure GelMA hydrogel, the degradation rate of GelMA / PEGDA hydrogel was significantly slowed down due to the addition of non-biodegradable PEGDA. After 14 days of PBS immersion, it still retained 80% of the matrix. After the addition of ppy, its stability was further increased. After 14 days of PBS immersion, the matrix it still retained could reach 82% ( Figure 2 J). This relatively slow degradation rate indicates that the GelMA / PEGDA / ppy hydrogel has good overall stability. It is expected to meet the stability and biodegradability required for simulating in vivo applications during the entire culture period, while being able to more effectively simulate the cell microenvironment.

[0084] Example 2 Effect of the system of Example 1 on the biocompatibility of stem cells

[0085] Step 1: Collect cells by trypsin digestion and encapsulate the cells with hydrogel, and prepare 3 hydrogels for each group; transfer the prepared composite conductive hydrogel encapsulating stem cells to the culture wells of the electrical stimulation device, and add 2 ml of culture medium to each well.

[0086] Step 2: Change the medium every 2 days. After 1, 4 and 7 days of culture, discard the medium, wash 3 times with PBS, prepare the live-dead staining solution according to the instructions, add 600 μL / well to each well, incubate at 37°C for 20 min, discard the staining solution, and wash again 3 times with PBS; observe under a laser confocal microscope, live cells appear green, dead cells appear red, and randomly select areas for image collection; with the help of Image J software, count the number of live and dead cells in each area, and calculate cell viability: Cell viability (%) = number of live cells / (number of live cells + number of dead cells) Determination results:

[0087] like Figure 3 A, The cells in all groups showed good survival status. Image J quantitative results showed that the survival rate of each group was above 80%, proving that the system had good biocompatibility.

[0088] Example 3 Effects of electrical stimulation parameters applied to cells by the system of Example 1 on cell proliferation

[0089] Step 1: Collect cells by trypsin digestion and encapsulate the cells with hydrogel, and prepare 3 hydrogels for each group; transfer the prepared composite conductive hydrogel encapsulating stem cells to the culture wells of the electrical stimulation device, and add 2 ml of culture medium to each well.

[0090] Step 2, apply electrical stimulation to the cells for 3 days to obtain the optimal voltage value. Start the exogenous microcurrent power supply, connect it to the electrical stimulation device, set the predetermined stimulation parameters, first set the electrical stimulation time to 20min, the stimulation voltage to 0, 1.5, 2, 2.5, 3, 3.5V, and after the settings are completed, let the stimulation device work, and electrically stimulate the conductive hydrogel encapsulating bone marrow mesenchymal stem cells to place the cells in an electrical microenvironment. After the stimulation is completed, remove the exogenous electrical stimulation device and replace the liquid. The optimal voltage is selected from the above parameters, the stimulation voltage is set to 3v, and the stimulation time is 0, 20, 40, 60, 80min. Select the optimal stimulation time, and finally obtain the optimal electrical stimulation parameters. After the stimulation is completed, perform the CCK-8 experiment to obtain the optimal stimulation parameters.

[0091] Step three: Stimulation voltage selection. The stimulation parameters for each group were set at 3V / 40min.

[0092] Step 4: The proliferation rate of each group under the optimal stimulation parameters. Each group was stimulated under the optimal parameters for 1, 3, 5, and 7 days and then CCK-8 was performed to obtain the proliferation rate of each group.

[0093] Measurement results: Figure 4 The results of A and 4B showed that each group had the highest proliferation rate under the parameter of 3V / 40min per day stimulation. Further continuous stimulation for 7 days found that the group containing 1% polypyrrole had the highest proliferation rate. Figure 4 In C, ES refers to electrical stimulation (3 V / 40 min per day), “-ES” refers to the group with electrical stimulation for 20 min (3 V / 40 min) per day, such as GelMA / PEGDA / 1ppy-ES; “-W / O ES” refers to the group without electrical stimulation, such as GelMA / PEGDA / 1ppy-W / O ES.

[0094] Example 4 Using the system of Example 1 to apply electrical stimulation to cells for osteogenic induction

[0095] Step 1: The composite conductive hydrogel encapsulates cells to form a hydrogel three-dimensional culture platform, and then transfers them to an electrical stimulation device for electrical stimulation. The stimulation parameters are 3V / 40min per day, so that the cells are in an electrical microenvironment. After the stimulation is completed, the exogenous electrical stimulation device is removed and the fluid is replaced.

[0096] Step 2: Apply electrical stimulation to the cells for 7 consecutive days to explore the effect of electrical signals on cell osteogenic differentiation.

[0097] Step three, osteogenic differentiation was determined by ALP kit method, Sirius red kit method, and immunofluorescence staining.

[0098] Measurement results:

[0099] The results of ALP are as follows Figure 5 As shown in (A, D), after 3 and 7 days of daily ES (electrical stimulation), the GelMA / PEGDA / 1ppy-ES group showed the highest ALP activity. Figure 5 B, 5E) showed that GelMA / PEGDA / ppy-ES exhibited the highest collagen expression at both days 7 and 14. Figure 5 In the table, ES refers to electrical stimulation (3 V / 40 min per day), and GelMA / PEGDA / ppy-ES refers to the group with electrical stimulation of 3 V / 40 min per day; Figure 5 GelMA, GelMA / PEGDA, and GelMA / PEGDA / ppy in D, 5E, and 5F all represent groups without electrical stimulation.

[0100] Immunofluorescence was performed on days 7 and 14 to observe the expression of osteogenic-related protein RUNX2. Figure 5 C, 5F) showed similar trends to ALP and collagen secretion tests.

[0101] The MSCs cultured in the GelMA / PEGDA / 1ppy-ES group had higher protein expression than the other groups. The above research results confirmed that the osteogenic differentiation of stem cells was significantly promoted under the three-dimensional electrical stimulation system, and the cell proliferation rate was further increased under the electrical microenvironment.

[0102] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. An in vitro three-dimensional cell electrical stimulation osteogenic induction system based on functionalized GelMA hydrogel, characterized in that: It includes a hydrogel three-dimensional culture platform formed by wrapping cells with a composite conductive hydrogel and an electrical stimulation device for providing electrical stimulation function to the hydrogel three-dimensional culture platform; The hydrogel three-dimensional culture platform is constructed by the following method: S1. Preparation of GelMA; S2, preparing ppy nanowires; S3, preparing PEGDA; S4. Preparation of hydrogel precursor solution: S4-1. Construction of hydrogel three-dimensional culture platform: S4-2, dissolving GelMA in the photoinitiator solution, then adding PEGDA, stirring until completely dissolved, then adding ppy nanowires, and ultrasonically dispersing to obtain a hydrogel precursor solution; S4-3. Adding cells for culture into a solution of a hydrogel precursor and irradiating the solution with ultraviolet light to solidify the hydrogel precursor into a composite conductive hydrogel and encapsulate the cells, thereby constructing the hydrogel three-dimensional culture platform.

2. The in vitro three-dimensional cell electrical stimulation osteogenic induction system based on functionalized GelMA hydrogel according to claim 1, characterized in that: Step S1 is specifically as follows: 10 g of type A gelatin solid powder was added to 100 mL of PBS solution, stirred at 60 °C for 2 h, 6 mL of methacrylic anhydride was added dropwise at a rate of 0.5 mL / min under light protection, and the reaction was allowed to proceed for 3 h. PBS solution preheated to 37 °C was added to terminate the reaction. The product was dialyzed in deionized water for 7 days using an 8K-14KDa dialysis bag, and the solution in the dialysis bag was freeze-dried to obtain GelMA.

3. The in vitro three-dimensional cell electrical stimulation osteogenic induction system based on functionalized GelMA hydrogel according to claim 1, characterized in that: Step S2 is specifically as follows: Take 0.1g of hexadecyltrimethylammonium bromide and add it to 30mL of 0.5M hydrochloric acid solution. After stirring until it is completely dissolved, 0.3mL of pyrrole monomer is added dropwise at a rate of 0.4mL / min. Stir evenly, add 30mL of 0.5M HCL solution containing 0.2g of ammonium persulfate, react at 4°C and 500rpm for 8 hours, centrifuge at 11000rpm, collect the solid product, rinse with deionized water and anhydrous ethanol in turn, and dry to obtain ppy nanowires.

4. The in vitro three-dimensional cell electrical stimulation osteogenic induction system based on functionalized GelMA hydrogel according to claim 1, characterized in that: Step S3 is specifically as follows: S3-1, take 10-40g of PEG solid with a molecular weight of 2000 and add it to 30-120mL of dichloromethane, stir until completely dissolved, add 0.2-0.8mL of triethylamine and 0.35-1.4mL of acryloyl chloride in sequence at 0-10°C, and then react under a nitrogen atmosphere for 24-96 hours; S3-2. After the reaction is completed, 10-40 mL of 1-4 mol / L potassium carbonate solution is added to the product, and the resulting reaction solution is transferred to a separatory funnel. After shaking, the reaction solution is allowed to stand for stratification. The dichloromethane phase reaction solution at the bottom is taken, and magnesium sulfate powder is added to remove water. The supernatant is collected by centrifugation and concentrated by rotary evaporation. Diethyl ether is added to the resulting concentrated solution until a white powder solid is precipitated. The concentrated solution is filtered and the collected white solid sample is vacuum dried at 20-40°C to constant weight to obtain a crude PEGDA product. S3-3. The crude PEGDA product was dialyzed in deionized water for 36-144 hours using a dialysis bag and freeze-dried to obtain purified PEGDA.

5. The in vitro three-dimensional cell electrical stimulation osteogenic induction system based on functionalized GelMA hydrogel according to claim 4, characterized in that: Step S3 is specifically as follows: S3-1, take 20g of PEG solid with a molecular weight of 2000 and add it to 60mL of dichloromethane, stir until completely dissolved, add 0.4mL of triethylamine and 0.7mL of acryloyl chloride in sequence at 4°C, and then react for 48 hours under a nitrogen atmosphere; S3-2. After the reaction is completed, 20 mL of 2 mol / L potassium carbonate solution is added to the product, and the resulting reaction solution is transferred to a separatory funnel. After shaking, the reaction solution is allowed to stand for stratification. The dichloromethane phase reaction solution at the bottom is taken, and magnesium sulfate powder is added several times to remove water. The supernatant is collected by centrifugation and concentrated by rotary evaporation. Ether is added to the resulting concentrated solution until a white powder solid is precipitated. The collected white solid sample is transferred to a 25°C vacuum drying oven and dried to constant weight to obtain a crude PEGDA product. S3-3, the crude PEGDA product was dialyzed in deionized water for 72 hours using a dialysis bag with a molecular weight cutoff of 2000Da, and freeze-dried at -20°C to obtain purified PEGDA.

6. The in vitro three-dimensional cell electrical stimulation osteoinduction system based on functionalized GelMA hydrogel according to claim 1, characterized in that: The photoinitiator solution is prepared by preparing a photoinitiator LAP and a PBS solution.

7. The in vitro three-dimensional cell electrical stimulation osteogenic induction system based on functionalized GelMA hydrogel according to claim 6, characterized in that: Step S4 is specifically as follows: S4-2, add 50-200 mg of photoinitiator LAP into 50-200 mL of PBS solution, stir at 40-60° C. in the dark for 0.5-2 hours to obtain a photoinitiator solution; Take 0.1875-0.75g of GelMA prepared in step S1 and add it to 1-5mL of photoinitiator solution, stir at 40-60°C until it is completely dissolved, then add 1wt%-2wt% of PEGDA prepared in step S3, stir for 1.5-6h, then add 0.5wt%-1.5wt% of ppy nanowires prepared in step S2, stir and react for 1.5-6h to obtain a hydrogel precursor solution; S4-3, adding cells for culture into the solution of hydrogel precursor, irradiating with 405nm ultraviolet light for 45-180s, so that the hydrogel precursor is solidified into a composite conductive hydrogel and wraps the cells, thereby constructing the hydrogel three-dimensional culture platform.

8. The in vitro three-dimensional cell electrical stimulation osteogenic induction system based on functionalized GelMA hydrogel according to claim 7, characterized in that: Step S4 is specifically as follows: S4-2, add 100 mg of photoinitiator LAP into 100 mL of PBS solution, stir at 50° C. in dark for 1 hour to obtain a photoinitiator solution; 0.375 g of GelMA prepared in step S1 was added to 2.5 mL of photoinitiator solution, and stirred at 50° C. until completely dissolved. Then, 1 wt% to 2 wt% of PEGDA prepared in step S3 was added, and after stirring for 3 h, 0.5 wt% to 1.5 wt% of ppy nanowires prepared in step S2 were added, and the mixture was stirred for reaction for 3 h to obtain a hydrogel precursor solution. S4-3. Add the cells to be cultured into the solution of the hydrogel precursor, and irradiate with 405nm ultraviolet light for 90s to solidify the hydrogel precursor into a composite conductive hydrogel and wrap the cells, thereby constructing the hydrogel three-dimensional culture platform.

9. The in vitro three-dimensional cell electrical stimulation osteoinduction system based on functionalized GelMA hydrogel according to any one of claims 1 to 8, characterized in that: The electrical stimulation device comprises a cell culture well plate, a positive electrode and a negative electrode arranged in the culture wells of the cell culture well plate, and an electrical stimulation host connected to the positive electrode and the negative electrode for providing an electrical stimulation function.

10. The in vitro three-dimensional cell electrical stimulation osteogenic induction system based on functionalized GelMA hydrogel according to claim 9, characterized in that: The electrical stimulation method of this system is: The hydrogel three-dimensional culture platform prepared in step S4 is transferred to the culture well of the cell culture well plate. After the culture well is washed once with culture medium, fresh complete culture medium is added to the culture well, and the well is placed in a 37°C incubator containing 5% CO2 for static culture. The culture medium is replaced every 2-3 days. During the culture process, the electric stimulation host applies electric stimulation to the hydrogel three-dimensional culture platform in the culture well through the positive electrode and the negative electrode. The voltage of the electric stimulation is 1-5V, and the time of the electric stimulation is 20-80min per day.

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