Microgel bio-ink, microgel scaffolds comprising neuro-vascular networks, and methods of making and using the same

Abstract

The application provides a microgel bio-ink, a microgel scaffold containing a nerve-vascular network and a preparation method and application thereof, and belongs to the technical field of biological medicine. The microgel bio-ink prepared by the application has a significant self-healing ability when printing, ensures high-precision extrusion molding, has excellent shear thinning ability, and the viscosity is significantly reduced with the increase of the shear rate; the microgel bio-ink keeps a solid structure at a physiological temperature and has temperature-sensitive stability; the self-healing effect is significant, the mechanical stability of the printed body is improved, and the microgel bio-ink has good printing extrusion and forming ability. The application realizes the composite printing of neuron-carrying microspheres and vascular endothelial cells, successfully constructs a microgel scaffold containing a nerve-vascular network, and can be used for simulating the microenvironment interaction mechanism in vivo and providing an integrated solution for complex tissue defect repair.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to microgel bioinks, microgel scaffolds containing neural-vascular networks, their preparation methods and applications. Background Technology

[0002] Tissue engineering research based on 3D bioprinting technology can construct relatively complex tissue structures. However, while single bio-inks can simulate macroscopic structures, they struggle to replicate the multi-scale heterogeneous microenvironment of the extracellular matrix. This directly leads to deficiencies in key functions such as mechanical conduction, neural innervation, and blood supply in printed tissues, making it difficult to meet the high-precision manufacturing requirements of complex tissue scaffolds. Therefore, a new method is urgently needed to promote the regeneration of neural-vascular networks within regenerating tissues, which is crucial for enhancing tissue activity and achieving effective functional integration. The development and application of composite bio-inks are key technologies for achieving functional breakthroughs and applications in bioprinting.

[0003] Hydrogel microspheres are hydrogel particles with a size in the micrometer range, typically 100–200 μm in diameter. Their preparation techniques are diverse, including batch emulsion, microfluidics, electrohydraulic spraying, and mechanical fragmentation. Hydrogel microspheres can be used individually, aggregate to form particulate microgels, or embedded within bulk hydrogels. Compared to traditional bulk hydrogels, hydrogel microspheres offer advantages such as small size, injectability, modularity, and porosity, exhibiting excellent potential for promoting oxygen diffusion and nutrient transport. They also enhance cell-cell and cell-matrix interactions, resulting in cells displaying higher differentiation potential, colony-forming ability, and stemness. Importantly, hydrogel microspheres can aggregate to form microgel bioinks, providing a novel ink form for 3D printing. Furthermore, conductive materials can facilitate intercellular electrical signal exchange, mimicking the physiological microenvironment of electroactive tissues and promoting cell proliferation and differentiation. However, the introduction of traditional conductive materials (such as carbon nanotubes and graphene) often leads to decreased mechanical properties or cytotoxic risks. Summary of the Invention

[0004] The purpose of this invention is to provide microgel bio-inks, microgel scaffolds containing neural-vascular networks, their preparation methods and applications, which can be used to address the shortcomings of current tissue engineering in the field of neural and vascular regeneration and improve tissue function after transplantation and repair.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for preparing microgel bio-ink, comprising the following steps:

[0007] S1. Adipose-derived mesenchymal stem cells were resuspended in a hydrogel solution containing methacrylamide gelatin and MXene. The resulting resuspension was used as the dispersion phase reagent, and mineral oil containing surfactants was used as the continuous phase reagent. Hydrogel microspheres were prepared by microfluidic chip.

[0008] S2. The hydrogel microspheres are cross-linked and cured by ultraviolet light to obtain cross-linked hydrogel microspheres;

[0009] S3. The cross-linked hydrogel microspheres are subjected to directional induction differentiation treatment, so that the adipose mesenchymal stem cells in the cross-linked hydrogel microspheres are induced to differentiate into neuronal cells and / or vascular endothelial cells, to obtain hydrogel microspheres containing neuronal cells and / or hydrogel microspheres containing vascular endothelial cells.

[0010] S4. Mix hydrogel microspheres containing neuronal cells and / or hydrogel microspheres containing vascular endothelial cells with a hydrogel solution containing methacrylamide gelatin and MXene to obtain microgel bio-ink.

[0011] Preferably, in the microgel bio-ink, the hydrogel microspheres containing neuronal cells and / or the hydrogel microspheres containing vascular endothelial cells are 3 to 8 parts by weight, and the hydrogel solution containing methacrylamide gelatin and MXene is 0.5 to 1.5 parts by weight.

[0012] Preferably, in the mineral oil, the surfactant is Span 80, and the volume percentage of the surfactant is 1-3%.

[0013] The flow rate of the dispersed phase reagent is 3~8 mL / h;

[0014] The flow rate of the continuous phase reagent is 0.5~1.5 mL / h;

[0015] The cross-linking and curing time is 50-70 seconds;

[0016] The intensity of the ultraviolet light is 50~150 mW / cm²;

[0017] The cross-linked hydrogel microspheres also include cleaning with a buffer solution after cross-linking and curing.

[0018] Preferably, the hydrogel solution containing methacrylamide gelatin and MXene is obtained by a preparation method comprising the following steps:

[0019] Methacrylated gelatin, buffer solution, and photoinitiator were mixed and dissolved to obtain a methacrylated gelatin solution;

[0020] A solution of methacrylamide gelatin was mixed with a solution of MXene to obtain a hydrogel solution containing methacrylamide gelatin and MXene.

[0021] Preferably, in the GelMA-MXene hydrogel solution, the final concentration of the methacrylamide gelatin is 5-15% by mass-volume percentage;

[0022] In the GelMA-MXene hydrogel solution, the concentration of MXene is 0.1~0.3 mg / mL.

[0023] The present invention also provides a microgel bio-ink prepared by the above preparation method.

[0024] This invention also provides a method for preparing a microgel scaffold, comprising the following steps:

[0025] Using the aforementioned microgel bio-ink as a raw material, 3D bioprinting was performed, and the microgel scaffold was obtained after UV cross-linking and curing.

[0026] This invention also provides a method for preparing a microgel scaffold comprising a neurovascular network, comprising the following steps:

[0027] Using the above-mentioned microgel bio-ink as raw material, 3D bioprinting was carried out. The printed products were then subjected to UV cross-linking curing and culture to obtain a microgel scaffold containing a neural-vascular network.

[0028] The microgel bio-ink contains both hydrogel microspheres containing neuronal cells and hydrogel microspheres containing vascular endothelial cells.

[0029] The present invention also provides a microgel scaffold containing a neural-vascular network prepared by the above preparation method.

[0030] This invention also provides the application of the above-mentioned microgel bioink, the microgel scaffold prepared by the above-mentioned preparation method, or the above-mentioned microgel scaffold containing a neural-vascular network in constructing in vitro biomimetic tissue models, high-throughput drug screening, preparing artificial implant materials, preparing organ-on-a-chip matrices, preparing bioelectronic interfaces, or constructing biomechanical research models.

[0031] The beneficial effects of this invention are:

[0032] The microgel bio-ink prepared by this invention exhibits significant self-healing ability during printing, ensuring high-precision extrusion molding, excellent shear dilution ability, and viscosity that decreases significantly with increasing shear rate; it maintains a solid structure at physiological temperature and possesses temperature-sensitive stability; its significant self-healing effect enhances the mechanical stability of the printed body, and it has good printing extrusion and molding capabilities.

[0033] This invention enables the composite printing of neuronal and vascular endothelial cell microspheres. The entire microgel scaffold prepared shows significant expression of βIII-Tubulin and CD31. A microgel scaffold containing a neural-vascular network has been successfully constructed, which can be used to simulate the interaction mechanism of the in vivo microenvironment and provide an integrated solution for the repair of complex tissue defects. Attached Figure Description

[0034] Figure 1 Material characterization of GelMA-MXene hydrogel. (A) Morphology of GelMA-MXene hydrogel; (B) Scanning electron microscopy; (C) Detection of conductivity; (D) Conductivity and resistivity of the hydrogel; (E) Performance of the hydrogel in LED lamp circuit. (****) P <0.0001);

[0035] Figure 2 For (A) the microfluidic platform for fabricating hydrogel microspheres; (B) the hydrogel microspheres generated in the microfluidic chip; (C) the particle size distribution of hydrogel microspheres in mineral oil and PBS solutions; (D) the particle size of hydrogel microspheres in mineral oil and PBS solutions;

[0036] Figure 3 (A) Liveness and death staining of ADSCs in hydrogel microspheres; (B) Survival activity of ADSCs in hydrogel microspheres; (C) Proliferation activity of ADSCs in hydrogel microspheres. (Gel represents GelMA microspheres, GM represents GelMA-MXene microspheres) (*) P <0.05,** P <0.01, **** P <0.0001);

[0037] Figure 4 (A) Expression of neural-related genes. (B) Neural-related immunofluorescence staining (Gel represents GelMA, GM represents GelMA-MXene) (*** P <0.001, **** P <0.0001 (Gel represents GelMA microspheres, GM represents GelMA-MXene microspheres);

[0038] Figure 5 (A) Expression of vascular-related genes. (B) Vascular-related immunofluorescence staining (Gel represents GelMA, GM represents GelMA-MXene) (*) P <0.05, ****P<0.0001 (Gel represents GelMA microspheres, GM represents GelMA-MXene microspheres);

[0039] Figure 6 Rheological testing of GelMA-MXene hydrogels and microgels. (A) Shear dilution capacity testing; (B) Temperature-sensitive behavior testing; (C) Self-healing ability testing. (GM represents GelMA-MXene);

[0040] Figure 7 For 3D bioprinting large-size microgel scaffolds. (A) 3D printing data model; (B) Printing process diagram; (C) Printed object image; (D) Microgel scaffold 4x magnification field of view; (E) Microgel scaffold 10x magnification field of view; (F) Scaffold can be removed after 7 days of in vitro culture;

[0041] Figure 8 This is a panoramic scan image of the microgel scaffold containing a neural-vascular network according to the present invention. Detailed Implementation

[0042] This invention provides a method for preparing microgel bio-ink, comprising the following steps:

[0043] S1. Adipose-derived mesenchymal stem cells were resuspended in a hydrogel solution containing methacrylamide gelatin and MXene. The resulting resuspension was used as the dispersion phase reagent, and mineral oil containing surfactants was used as the continuous phase reagent. Hydrogel microspheres were prepared by microfluidic chip.

[0044] S2. The hydrogel microspheres are cross-linked and cured by ultraviolet light to obtain cross-linked hydrogel microspheres;

[0045] S3. The cross-linked hydrogel microspheres are subjected to directional induction differentiation treatment, so that the adipose mesenchymal stem cells in the cross-linked hydrogel microspheres are induced to differentiate into neurons and / or vascular endothelial cells, to obtain hydrogel microspheres containing neurons and / or hydrogel microspheres containing vascular endothelial cells.

[0046] S4. Hydrogel microspheres containing neuronal cells and / or hydrogel microspheres containing vascular endothelial cells are mixed with a hydrogel solution containing methacrylamide gelatin and MXene to obtain a microgel bio-ink. Preferably, in the microgel bio-ink, the hydrogel microspheres containing neuronal cells and / or hydrogel microspheres containing vascular endothelial cells, by weight, are 3-8 parts, and the hydrogel solution containing methacrylamide gelatin and MXene, by weight, is 0.5-1.5 parts. Preferably, in the mineral oil, the surfactant is Span 80, and the volume percentage of the surfactant is 1-3%; the flow rate of the dispersed phase reagent is 3-8 mL / h; the flow rate of the continuous phase reagent is 0.5-1.5 mL / h; the crosslinking and curing time is 50-70 s; the ultraviolet light intensity is 50-150 mW / cm²; the crosslinked hydrogel microspheres are further washed with a buffer solution after crosslinking and curing. Preferably, the hydrogel solution containing methacrylamide gelatin and MXene is obtained by a preparation method comprising the following steps: mixing and dissolving methacrylamide gelatin, buffer solution, and photoinitiator to obtain a methacrylamide gelatin solution; mixing the methacrylamide gelatin solution with an MXene solution to obtain a hydrogel solution containing methacrylamide gelatin and MXene. Preferably, in the GelMA-MXene hydrogel solution, the final concentration of methacrylamide gelatin, by mass-volume percentage, is 5-15%; and the concentration of MXene in the GelMA-MXene hydrogel solution is 0.1-0.3 mg / mL.

[0047] The present invention also provides a microgel bio-ink prepared by the above preparation method.

[0048] This invention also provides a method for preparing a microgel scaffold, comprising the following steps:

[0049] Using the aforementioned microgel bio-ink as a raw material, 3D bioprinting was performed, and the microgel scaffold was obtained after UV cross-linking and curing.

[0050] This invention also provides a method for preparing a microgel scaffold comprising a neurovascular network, comprising the following steps:

[0051] Using the above-mentioned microgel bio-ink as raw material, 3D bioprinting was carried out. The printed products were then subjected to UV cross-linking curing and culture to obtain a microgel scaffold containing a neural-vascular network.

[0052] The microgel bio-ink contains both hydrogel microspheres containing neuronal cells and hydrogel microspheres containing vascular endothelial cells; the culture is preferably carried out using a mixed culture medium containing vascular endothelial induction medium and neuroinduction medium.

[0053] The present invention also provides a microgel scaffold containing a neural-vascular network prepared by the above preparation method.

[0054] This invention also provides the application of the above-mentioned microgel bio-ink, the microgel scaffold prepared by the above-mentioned preparation method, or the above-mentioned microgel scaffold containing a neural-vascular network in constructing in vitro biomimetic tissue models, high-throughput drug screening, preparation of artificial implant materials, preparation of organ-on-a-chip substrates, preparation of bioelectronic interfaces, or construction of biomechanical research models; the construction of in vitro biomimetic tissue models includes simulating the physiological microenvironment based on neural-vascular networks to support the study of complex tissue functions; the high-throughput drug screening includes evaluating the permeability, toxicity, and efficacy of neuro / vascular targeted drugs through the interaction between neural-vascular networks and drugs; the artificial implant materials include promoting the regeneration and functional reconstruction of damaged nerves / blood vessels; the organ-on-a-chip substrate includes constructing a chip system containing a neural-vascular interface to simulate organ-level physiological / pathological processes; the preparation of bioelectronic interfaces includes combining their conductivity and biocompatibility to develop implantable bioelectrodes or neural signal recording devices; and the construction of biomechanical research models includes simulating the in vivo mechanical microenvironment to study the growth and adaptability of neural / vascular tissues under stress.

[0055] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0056] Example

[0057] (I) Material Synthesis

[0058] 1. Synthesis and Characterization of GelMA-MXene

[0059] Weigh 1 g of dried, white, foamy GelMA and add it to a centrifuge tube. Then, add 9 mL of PBS buffer and 1 mL of 2.5% LAP photoinitiator to the centrifuge tube. Place the centrifuge tube in a 50°C oven and dissolve in the dark for 1 hour, shaking frequently to ensure complete dissolution. A 10% (w / v) GelMA solution is obtained after dissolution. Next, the hydrogel solution is filtered through a 0.22 μm filter under light-protected conditions to obtain a sterile GelMA hydrogel solution.

[0060] MXene (25 mg / mL) (Beike Nano, BK2020040305, China) was sterilized by cobalt-60 irradiation. Then, 80 μL of the MXene solution was added to 10 mL of GelMA solution, and the mixture was repeatedly pipetted to ensure thorough mixing of MXene with the hydrogel, yielding a GelMA-MXene hydrogel solution. The GelMA concentration was 10% (w / v), and the MXene concentration was 0.2 mg / mL. The prepared GelMA-MXene hydrogel was translucent black. Figure 1 A).

[0061] 2. Scanning electron microscope

[0062] GelMA-MXene hydrogels were cross-linked to prepare cylindrical samples, which were then frozen at -20℃ and -80℃ for one day each. The samples were then freeze-dried in a vacuum freeze dryer for one day. After removal, the samples were subjected to brittle fracture in liquid nitrogen to obtain the hydrogel cross-section. After gold sputtering, the internal structure of the GelMA-MXene hydrogel was observed using scanning electron microscopy, revealing that MXene particles were uniformly distributed within the hydrogel. Figure 1 B).

[0063] 3. Conductivity detection of GelMA-MXene hydrogel

[0064] 200 μL of GelMA hydrogel solution and GelMA-MXene hydrogel solution were respectively prepared into cylindrical samples and placed on a crosslinking four-probe tester to ensure that the probes were in full contact with the hydrogels. The resistivity of the samples was then measured. Figure 1 C). The conductivity is calculated using the conductivity formula: resistivity ρ = 1 / conductivity σ.

[0065] Conductivity testing showed that the conductivity of the GelMA-MXene hydrogel was (1.74 ± 0.09 S / m), significantly higher than that of the pure GelMA hydrogel (1.33 ± 0.10 S / m). P <0.0001). Correspondingly, the resistivity of the GelMA-MXene hydrogel is (0.58 ± 0.03 Ω·m), which is lower than that of the pure GelMA hydrogel (0.75 ± 0.06 Ω·m). P <0.0001)( Figure 1 D).

[0066] GelMA-MXene hydrogel and pure GelMA hydrogel were respectively connected to a circuit consisting of an LED light, a battery, and wires. The results showed that both hydrogel materials allowed current to pass through, thus connecting the circuit and lighting the LED light. The LED light lit by the GelMA-MXene hydrogel was brighter, further demonstrating its superior conductivity. Figure 1 E).

[0067] (II) Fabrication of hydrogel microspheres

[0068] 1. Method for preparing hydrogel microspheres

[0069] ADSCs from generations P3 to P5 were digested with 0.25% trypsin and centrifuged, then counted to obtain 1×10⁻⁶ cells. 7 Each cell was collected. The supernatant was then removed by centrifugation, retaining the cell pellet. The cells were then resuspended in 1 mL of GelMA-MXene hydrogel solution. Next, the GelMA-MXene hydrogel solution containing the ADSCs cells was transferred to a 1 mL sterile syringe. Simultaneously, 5 mL of mineral oil containing 2% Span 80 was drawn using a 5 mL sterile syringe.

[0070] Before the experiment, the silicone tubing, microfluidic chip surface, and internal channels were rinsed and disinfected with 75% alcohol, and then placed in a clean bench for 1 hour of UV light sterilization. Syringes containing hydrogel and mineral oil were respectively installed on a dual-channel microinjection pump, with flow rates set to 5 mL / h and 1 mL / h, respectively. Subsequently, cell-loaded GelMA-MXene-ADSCs hydrogel microspheres were prepared under aseptic conditions.

[0071] Utilizing the high flow rate of mineral oil, the oil-water immiscibility principle, and the surface tension of surfactants, the hydrogel solution was sheared into uniformly sized hydrogel microspheres within a microfluidic chip. The hydrogel microspheres flowed out from the microfluidic chip's outlet channel, were irradiated with 405 nm ultraviolet light for cross-linking and curing for approximately 60 seconds, with the ultraviolet light intensity around 100 mW / cm². The cross-linked hydrogel microspheres were collected using centrifuge tubes and stabilized in ice water. Figure 2 A, B).

[0072] 2. Cleaning of hydrogel microspheres

[0073] After preparation, aspirate and discard the upper layer of mineral oil. Then add PBS buffer and repeatedly pipette to wash away any remaining mineral oil on the surface of the hydrogel microspheres. Next, centrifuge the tube at 1200 rpm for 5 minutes. After centrifugation, discard the upper layer of mineral oil and the middle layer of PBS buffer, transfer the remaining hydrogel microspheres to a new centrifuge tube, add PBS buffer, and pipette again. Then, centrifuge again to further remove any remaining mineral oil on the surface of the hydrogel microspheres. Repeat this washing step 2-3 times until clean hydrogel microspheres are obtained.

[0074] By observing the morphology of hydrogel microspheres in mineral oil and PBS buffer solutions, this invention found that the hydrogel microspheres were uniform in size and shape in both solutions. However, the particle size of the microspheres in the PBS buffer solution was significantly larger than that in the mineral oil solution, indicating that the microspheres exhibited significant water absorption and swelling after transferring from the oil phase to the aqueous phase. Figure 2 C). The particle size of the GelMA-MXene hydrogel microspheres in mineral oil was 166.4 ± 2.62 μm, and the particle size after transfer to PBS buffer was 205.2 ± 9.54 μm. Figure 2 D). Moreover, the microspheres exhibit uniform particle size distribution in different liquid environments, showing a normal distribution. Figure 3-5 B).

[0075] 3. Activity detection of ADSCs in hydrogel microspheres

[0076] 3.1 Live and dead staining of ADSCs in hydrogel microspheres

[0077] GelMA-MXene-ADSCs hydrogel microspheres were added to 48-well plates, 50 μL of microspheres per well, along with 200 μL of DMEM complete medium. The plates were incubated at 37°C in a 5% CO2 incubator. Cell viability and mortality staining was performed on days 1, 4, and 7 of culture. After staining, the microspheres were transferred to small dishes, and the morphology of the microspheres and cells was observed using a laser confocal microscope. Cell viability was calculated by dividing the number of viable cells by the total number of cells. The control group consisted of GelMA-ADSCs hydrogel microspheres.

[0078] On days 1, 4, and 7 of culture, the cell viability in GelMA-MXene hydrogel microspheres was higher than that in GelMA hydrogel microspheres. Specifically, the cell viability rates were (63.92 ± 3.08% and 58.85 ± 1.0%), (89.63 ± 1.41% and 84.19 ± 2.23%), and (95.39 ± 1.15% and 90.39 ± 1.71%), respectively, and these differences were statistically significant (*). P<0.05)( Figure 3 A).

[0079] 3.2 Proliferation activity of ADSCs in hydrogel microspheres

[0080] GelMA-MXene-ADSCs hydrogel microspheres were added to 48-well plates, 50 μL of microspheres to each well, along with 200 μL of DMEM complete medium. The plates were incubated at 37°C in a 5% CO2 incubator. Cell proliferation was assessed using a CCK-8 assay on days 1, 4, and 7. After incubation, 100 μL of the supernatant from each well was transferred to a 96-well plate. The absorbance of the 96-well plate was measured at 450 nm using a multi-plate reader. The optical density (OD value) of the absorbance was directly proportional to the number of viable cells. Three 48-well replicates were set up for each group. The control group also consisted of GelMA-ADSCs hydrogel microspheres.

[0081] On days 4 and 7 of culture, the proliferation activity of ADSCs in GelMA-MXene hydrogel microspheres was significantly higher than that in GelMA hydrogel microspheres. P <0.01, **** P <0.0001)( Figure 3 C). This indicates that GelMA-MXene hydrogel microspheres have better performance in supporting cell survival and proliferation.

[0082] (III) Directional Differentiation Induction of GelMA-MXene-ADSCs Hydrogel Microspheres

[0083] 1. GelMA-MXene hydrogel microspheres induce ADSCs to differentiate into neural cells.

[0084] The prepared 1 mL GelMA-MXene-ADSCs hydrogel microspheres were cultured in DMEM complete medium for 2 days. Then the old medium was removed, the microspheres were washed, and neural induction medium was added.

[0085] The neural induction protocol was as follows: (1) First, 1 mL of neural pre-induction medium was added to each well. This medium consisted of Neurobasal medium, 2% B27, 20 ng / mL EGF, 10 ng / mL FGF, and 1% penicillin / streptomycin solution. (2) After 1 day, the pre-induction medium was removed and replaced with neural induction medium, namely Neurobasal medium with 2% B27 and 1% penicillin / streptomycin solution. Thereafter, the medium was changed every 2 days, and induction culture was performed for 4, 7, and 10 days, respectively. The control group consisted of GelMA-ADSCs hydrogel microspheres.

[0086] 2. GelMA-MXene hydrogel microspheres induce ADSCs to differentiate into vascular endothelial cells.

[0087] The prepared 1 mL GelMA-MXene-ADSCs hydrogel microspheres were cultured in DMEM complete medium for 2 days. Then the old medium was removed, the microspheres were washed, and neural induction medium was added.

[0088] The angiogenesis induction protocol was as follows: endothelial cell culture medium was used, supplemented with 50 ng / mL VEGF, 10 ng / mL FGF, and 1% penicillin / streptomycin solution. The culture medium was changed daily, and induction was performed for 4, 7, and 10 days, respectively. The control group consisted of GelMA-ADSCs hydrogel microspheres.

[0089] 3. qPCR detection of the differentiation-inducing effect of cell-loaded hydrogel microspheres

[0090] On days 4, 7, and 10 of induction, cell-loaded hydrogel microspheres were collected from 12-well plates and transferred to centrifuge tubes. GelMA lysis buffer was added, and the tubes were incubated at 37°C for 30 minutes to ensure complete lysis of the hydrogel microspheres and release the cells. After incubation, the cells were centrifuged at 1200 rpm to remove the supernatant, retaining the cell pellet. RNA was then extracted and reverse transcribed, followed by qPCR experiments. Finally, the expression levels of neurally related genes (TUBB3, MAP2) and vascular-related genes (KDR, ANGPT) were analyzed.

[0091] 4. Immunofluorescence staining to assess the differentiation-inducing effect of cell-loaded hydrogel microspheres

[0092] On day 7 of induction, the induced cell-carrying hydrogel microspheres were collected into centrifuge tubes. Subsequently, the microspheres were fixed with 4% paraformaldehyde solution for 24 hours. After fixation, permeabilization and blocking treatments were performed. βIII-Tubulin and MAP2 were used as neuro-associated antibodies, and CD31 and VEGF-1 were used as vascular-associated antibodies for immunofluorescence staining of the hydrogel microspheres. The treated microspheres were then added to confocal microscopy dishes, and images were acquired and observed using a laser confocal microscope.

[0093] Neural induction experiments revealed no significant difference in the relative expression levels of two neural genes between GelMA-MXene and GelMA microspheres on day 1. However, on day 7, the relative expression levels of neural-related genes (TUBB3 and MAP23) in GelMA-MXene hydrogel microspheres were significantly higher than those in GelMA hydrogel microspheres. P<0.0001). On day 10, the relative expression level of TUBB3 in GelMA-MXene hydrogel microspheres was higher than that in GelMA hydrogel microspheres, and the difference was statistically significant (****). P <0.0001, *** P <0.001). Furthermore, on day 7, the expression levels of neural-related genes in the GelMA-MXene hydrogel microspheres were higher than at other time points, indicating that the differentiation induction effect was best on day 7. Figure 4 A).

[0094] Therefore, on day 7 of neural induction culture, the hydrogel microspheres were selected for immunofluorescence staining. The results showed that cells in both groups of hydrogel microspheres expressed the neuron-associated antibodies βIII-Tubulin (labeling neuronal axons) and MAP2 (labeling neuronal dendrites and cell bodies), indicating that ADSCs had been successfully induced to differentiate into neurons within the hydrogel microspheres. Figure 4 B).

[0095] The angiogenesis induction assay showed that on day 1, the relative expression levels of vascular-related genes (KDR and ANGPT1) in GelMA-MXene hydrogel microspheres were higher than those in GelMA hydrogel microspheres, and the difference was statistically significant (*). P <0.05). On day 7, the relative expression levels of KDR and ANGPT1 in GelMA-MXene hydrogel microspheres were also higher than those in GelMA hydrogel microspheres, and the differences were statistically significant (****). P <0.0001). On day 10, there was no significant difference in the relative expression levels of the two vascular genes between GelMA-MXene and GelMA microspheres. Furthermore, on day 7, the expression levels of vascular-related genes in GelMA-MXene hydrogel microspheres were higher than at other time points, indicating that the differentiation induction was most effective on day 7. Figure 5 A).

[0096] Therefore, on day 7 of vascular induction culture, the hydrogel microspheres were selected for immunofluorescence staining. The results showed that cells in both groups of hydrogel microspheres expressed vascular-associated antibodies CD31 and VEGF1 (labeling vascular endothelial cells), indicating that ADSCs had been successfully induced to differentiate into vascular endothelial cells in the hydrogel microspheres. Figure 5 B).

[0097] (iv) Microgel preparation and characterization

[0098] 1. Microgel preparation

[0099] The hydrogel microspheres were transferred to centrifuge tubes and centrifuged at 1200 rpm for 5 minutes using a high-speed centrifuge. The supernatant aqueous solution was then discarded. Next, the microspheres were mixed with GelMA-MXene hydrogel solution at a ratio of 5:1 and centrifuged again to allow the hydrogel solution to replace the water in the gaps between the microspheres. This process was repeated 2-3 times to finally obtain a microgel bio-ink with a microsphere to hydrogel solution ratio of 5:1.

[0100] 2. Rheological detection of microgels

[0101] Take 150 μl of GelMA-MXene microgel and place it on the rheometer platform. Select an SP25 test rotor with a diameter of 25 mm and set the measurement gap to 0.2 mm.

[0102] (1) Shear dilution capacity test: The relationship between shear rate and viscosity was determined in the range of 0.1~1000 / s at 25°C to evaluate the shear dilution capacity of the microgel. (2) Temperature-sensitive behavior test: The storage modulus (G') and loss modulus (G”) of the microgel were measured under temperature changes from 37°C to 4°C (strain parameter: 1%, frequency parameter: 1 Hz) to study the temperature-sensitive behavior of the microgel. (3) Self-healing ability test: The self-healing ability of the microgel was evaluated by alternating low strain (strain 1%, frequency 1 Hz) and high strain (strain 300%, frequency 1 Hz) at 20°C.

[0103] Rheological analysis of the microgels revealed that both GelMA-Mxene microgels and hydrogels exhibited good shear-dilution properties, meaning their viscosity gradually decreased with increasing shear rate. Furthermore, the microgels showed higher viscosity than the hydrogels, indicating that the better structural stability maintained by the microgels during printing is crucial for 3D extrusion printing. Figure 6 A).

[0104] Temperature measurement results further demonstrate the superior performance of the microgels. At temperatures below 20°C, the storage modulus of the GelMA-Mxene hydrogel begins to exceed its loss modulus, and its complex viscosity also increases, indicating a transition from a liquid to a solid state. The microgels, however, exhibit more stable properties; their storage modulus consistently exceeds their loss modulus and remains unaffected by temperature changes. This further demonstrates the greater stability of the microgels and the wider temperature window that can be set during 3D printing operations. Figure 6 B).

[0105] The self-healing ability test results showed that both groups of samples exhibited self-healing phenomena during multiple alternating cycles. Notably, the healing curve of the microgel was more regular and flatter than that of the control group, indicating a more significant self-healing effect. Figure 6 C).

[0106] 3. Microgel 3D Printing Experiment

[0107] After the microgel was prepared, it was transferred to a 5 mL syringe using a pipette, and excess air bubbles were removed. Printing experiments were conducted using a 3D BioMaker4 bioprinter. The printing temperature was 20℃, the printing platform temperature was 4℃, the nozzle printing speed was 2 mm / s, the extrusion speed was 0.3 mm³ / s, the retraction distance was 0.1 mm, and the needle lift-off distance after printing was 1 mm. A 5 mL syringe was used as the printing cartridge during the printing process, and the syringe needle was a G23 specification with an inner diameter of 0.34 mm.

[0108] Next, the syringe was placed in the printer barrel and pre-cooled for 10 minutes before 3D printing. After printing, the scaffold was placed under 405 nm ultraviolet light for 1 minute for cross-linking, with the light intensity set to 100 mW / cm². Finally, the resolution and microstructure of the printed body were observed to evaluate the printing effect.

[0109] The microgel was used as a 3D printing ink in printing tests, and the ink was found to have good printing extrusion and forming capabilities. A large-sized cell-loaded scaffold (2×2 cm) was successfully printed. This scaffold not only exhibited good printing ability but also had a clear structure and smooth lines, fully demonstrating the excellent printing performance of the microgel bio-ink. Microscopic observation showed that cells were evenly distributed within the microgel scaffold. More importantly, after culturing the scaffold in culture medium for 7 days, the scaffold not only maintained its initial shape stability without significant deformation or collapse, but also retained its structural integrity. After culturing, the scaffold could be easily removed without fragmentation or damage, further verifying its good mechanical properties and stability. Figure 7 ).

[0110] 4. 3D-printed neurovascular network microgel scaffold

[0111] 500 μL each of neurogenic and vascularized microspheres were collected on day 7 of induced differentiation. The two types of microspheres were first mixed in a 1:1 ratio, and then the total amount of microspheres was mixed with GelMA-MXene hydrogel solution in a 5:1 ratio to prepare a microgel. Subsequently, a 2×2 cm neurovascular microgel scaffold was 3D bioprinted into a confocal dish. Endothelial induction medium and neural induction medium were mixed in a 1:1 ratio, and 3 mL of the mixed induction medium was added to the dish to submerge the scaffold. The microgel scaffold was then cultured.

[0112] After 3 days of culture, the culture medium was removed and the microgel scaffolds were washed twice with PBS buffer, then fixed with 4% paraformaldehyde. Following fixation, permeabilization and blocking were performed. βIII-Tubulin was used as the neuro-associated antibody, and CD31 as the vascular-associated antibody for immunofluorescence staining of the microgel scaffolds. Images were observed and acquired using a laser confocal microscope.

[0113] Immunofluorescence staining results after 3 and 7 days of post-printing culture showed that the neural marker βIII-Tubulin and the vascular marker CD31 were both significantly expressed and uniformly distributed within the constructed microgel scaffold. Panoramic scanning images further confirmed this, showing significant expression of βIII-Tubulin and CD31 throughout the entire microgel scaffold, indicating that this invention successfully constructed a microgel scaffold containing a neural-vascular network. Figure 8 ).

[0114] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a microgel bio-ink, characterized in that, Includes the following steps: S1. Adipose-derived mesenchymal stem cells were resuspended in a hydrogel solution containing methacrylamide gelatin and MXene. The resulting resuspension was used as the dispersion phase reagent, and mineral oil containing surfactants was used as the continuous phase reagent. Hydrogel microspheres were prepared by microfluidic chip. S2. The hydrogel microspheres are cross-linked and cured by ultraviolet light to obtain cross-linked hydrogel microspheres; S3. The cross-linked hydrogel microspheres are subjected to directional induction differentiation treatment, so that the adipose mesenchymal stem cells in the cross-linked hydrogel microspheres are induced to differentiate into neurons and vascular endothelial cells, to obtain hydrogel microspheres containing neurons and hydrogel microspheres containing vascular endothelial cells. S4. Hydrogel microspheres containing neuronal cells and hydrogel microspheres containing vascular endothelial cells are mixed with a hydrogel solution containing methacrylamide gelatin and MXene to obtain microgel bio-ink. In the mineral oil, the surfactant is Span 80, and the volume percentage of the surfactant is 1-3%. The flow rate of the dispersed phase reagent is 3~8 mL / h; The flow rate of the continuous phase reagent is 0.5~1.5 mL / h; The cross-linking and curing time is 50-70 seconds; The intensity of the ultraviolet light is 50~150 mW / cm². 2 ; The hydrogel solution containing methacrylamide gelatin and MXene is obtained by a preparation method including the following steps: Methacrylated gelatin, buffer solution, and photoinitiator were mixed and dissolved to obtain a methacrylated gelatin solution; A solution of methacrylamide gelatin was mixed with a solution of MXene to obtain a hydrogel solution containing methacrylamide gelatin and MXene. In the hydrogel solution containing methacrylamide gelatin and MXene, the final concentration of methacrylamide gelatin is 5-15% by mass-volume percentage. In the hydrogel solution containing methacrylamide gelatin and MXene, the concentration of MXene is 0.1~0.3 mg / mL.

2. The preparation method according to claim 1, characterized in that, In the microgel bio-ink, the hydrogel microspheres containing neuronal cells and the hydrogel microspheres containing vascular endothelial cells are 3 to 8 parts by weight, and the hydrogel solution containing methacrylamide gelatin and MXene is 0.5 to 1.5 parts by weight.

3. The preparation method according to claim 1, characterized in that, The cross-linked hydrogel microspheres also include cleaning with a buffer solution after cross-linking and curing.

4. Microgel bio-ink prepared by the preparation method according to any one of claims 1 to 3.

5. A method for preparing a microgel scaffold, characterized in that, Includes the following steps: Using the microgel bio-ink described in claim 4 as a raw material for 3D bioprinting, a microgel scaffold is obtained after ultraviolet light crosslinking and curing.

6. A method for preparing a microgel scaffold comprising a neural-vascular network, characterized in that, Includes the following steps: Using the microgel bio-ink described in claim 4 as a raw material for 3D bioprinting, the printed product is sequentially subjected to ultraviolet light crosslinking curing and culture to obtain a microgel scaffold containing a neural-vascular network. The microgel bio-ink contains both hydrogel microspheres containing neuronal cells and hydrogel microspheres containing vascular endothelial cells.

7. A microgel scaffold containing a neural-vascular network prepared by the preparation method of claim 6.

8. The application of the microgel bio-ink of claim 4, the microgel scaffold prepared by the preparation method of claim 5, or the microgel scaffold containing a neural-vascular network of claim 7 in constructing in vitro biomimetic tissue models, high-throughput drug screening, preparing artificial implant materials, preparing organ-on-a-chip matrices, preparing bioelectronic interfaces, or constructing biomechanical research models.

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