3D bio-printed vascularization-promoting bladder patch and preparation method thereof

Abstract

The invention belongs to the field of biomedicine, and particularly relates to a 3D bio-printed vascularization-promoting bladder patch and a preparation method thereof. The preparation method comprises the following steps: polymerizing prepared GelMA and Laponite to prepare GelMA-Laponite hydrogel, then mixing and printing the GelMA-Laponite hydrogel and seed cells on a bladder acellular matrix by using a 3D biological printing machine to form a GelMA-Laponite stent with a three-dimensional space structure, and crosslinking the stent by using ultraviolet light to obtain the double-layer vascularized bladder patch. The construction level of the tissue-engineered bladder patch is expected to be improved, the vascularization problem of the tissue-engineered bladder graft is solved, and a good theoretical basis and technical support are laid for promoting the clinical application process of bladder tissue engineering.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a 3D bioprinted vascularization-promoting bladder patch and a preparation method thereof. Background Art

[0002] Congenital and acquired bladder diseases (such as bladder exstrophy, neurogenic bladder, malignant tumors and trauma) may lead to anatomical or functional defects of the bladder, often requiring surgery to protect kidney function. Severe cases may even require radical cystectomy and urinary diversion, which seriously affects the patient's quality of life.

[0003] In recent years, with the innovation and development of tissue engineering technology, the construction of functional bionic bladder tissue in vitro to repair or replace damaged bladders has opened up a new way for tissue function repair, reconstruction and replacement therapy after bladder injury, and has broad prospects for clinical application. Current studies have reported a variety of scaffold materials with high mechanical strength, good biocompatibility and good cell adhesion, screened out seed cells such as autologous cells and stem cells that can be used for bladder tissue construction, and constructed a series of single-layer or multi-layer, cell-free or cell-containing bionic bladder tissues. However, the engineered bladder tissue constructed using existing biomaterials and bionic construction strategies is quite different from normal bladder tissue in terms of structure and function, and there are still many deficiencies in integration with the host after transplantation and bladder repair effect. Among them, the vascularization problem of tissue engineering bladder transplants is the main technical barrier hindering the clinical application transformation of this technology.

[0004] Blood vessels are a key component of the tissue microenvironment. Traditionally, the role of the vascular system is to provide nutrients and oxygen to surrounding cells, but it is now also recognized that the vascular system can provide signal cues that affect the biological outcomes of regeneration and disease. Therefore, excellent vascularization performance is a necessary condition to ensure that tissue-engineered tissues survive and function in vivo.

[0005] 3D bioprinting technology is an emerging biomedical technology that can achieve precise printing control of tissue structures. It provides a new method for regenerative medicine and tissue engineering by manufacturing 3D cell-loaded stereoscopic structures in vitro. In recent years, it has also been used in a variety of applications in organ reconstruction and repair tissue regeneration. In the bioprinting process, a biomaterial solution or a hydrogel mixture of several biomaterials that encapsulates cells is called bioink. An ideal bioink should have good rheological properties and biological properties, as well as mechanical properties. Bioinks should be cross-linked or stabilized during or immediately after bioprinting to generate the final shape and structure of the designed structure. At present, there are many types of bioinks used in 3D bioprinting with different functions, but the common disadvantage is that the fidelity of the printed structure is poor. Summary of the invention

[0006] The purpose of the present invention is to provide a biological ink.

[0007] Another object of the present invention is to provide a method for preparing the above-mentioned biological ink

[0008] Another object of the present invention is to provide a 3D printed biological bladder patch.

[0009] Another object of the present invention is to provide a method for preparing the above-mentioned bladder patch.

[0010] According to the bio-ink of a specific embodiment of the present invention, the bio-ink is a composite hydrogel solution made of methacrylic anhydride gelatin solution and Laponite dispersion, wherein:

[0011] The concentration of methacrylic anhydride gelatin is 5 to 15% (w / v).

[0012] The concentration of Laponite is 0.2 to 2% (w / v).

[0013] According to the biological ink of a specific embodiment of the present invention, the methacrylic anhydride gelatin solution is obtained by dissolving freeze-dried methacrylic anhydride gelatin in a buffer solution, preferably, the dissolution is carried out at 30-40°C, and particularly preferably, the freeze-dried methacrylic anhydride gelatin is dissolved in a buffer solution at 37°C to obtain the methacrylic anhydride gelatin solution.

[0014] The buffer solution includes one or more of phosphate buffer solution, Tris buffer solution, and borate buffer solution.

[0015] According to the bio-ink of a specific embodiment of the present invention, the Laponite dispersion is obtained by dispersing Laponite powder in deionized water and stirring.

[0016] Laponite dispersion can be mixed with methacrylic anhydride gelatin solution to obtain a composite hydrogel solution, while Laponite cannot be directly dissolved in methacrylic anhydride gelatin.

[0017] According to a specific embodiment of the present invention, the method for preparing biological ink comprises the following steps:

[0018] The methacrylic anhydride gelatin solution is mixed with the Laponite dispersion and stirred to obtain a composite hydrogel solution, which is the bio-ink.

[0019] According to a specific embodiment of the present invention, the 3D printed biological bladder patch comprises a bladder decellularized matrix and a 3D printed scaffold, and the 3D printed scaffold is 3D printed on the bladder decellularized matrix by the biological ink.

[0020] According to a specific embodiment of the present invention, a method for preparing a 3D printed biological bladder patch comprises the following steps:

[0021] (1) placing the bladder decellularized matrix in a 3D bioprinter, with the muscle layer of the bladder decellularized matrix facing upward;

[0022] (2) 3D printing of cell-laden bio-ink onto a bladder decellularized matrix;

[0023] (3) After printing is completed, the printed material is cross-linked with ultraviolet light and incubated.

[0024] The bio-ink contains adipose-derived stem cells (ADSCs) at a concentration of 1x10 7 / mL. Adipose-derived stem cells (ADSCs) were preferably selected. The p3-p6 cells were digested with trypsin and centrifuged. The supernatant was aspirated and evenly resuspended with a certain amount of composite hydrogels of different concentrations to obtain a bio-ink containing cells, wherein the concentration of adipose-derived stem cells was 1x10 7 / mL.

[0025] According to the method for preparing a 3D printed biological bladder patch according to a specific embodiment of the present invention, in step (2), the 3D printing parameters are that the syringe temperature is set to 20-25° C., and / or,

[0026] The platform temperature is 3-6°C, and / or,

[0027] Printing speed is 1.5-2.5mm / s, and / or,

[0028] Extrusion rate is 0.25-0.35mm 3 / s.

[0029] According to the method for preparing a 3D printed biological bladder patch according to a specific embodiment of the present invention, in step (3), the printed material is irradiated with 405 nm ultraviolet light for 40 seconds at a light intensity of 80 mW / cm 2 .

[0030] According to the method for preparing a 3D printed biological bladder patch according to a specific embodiment of the present invention, in step (3), after the printed material is cross-linked by ultraviolet light, DMEM culture medium and 10% fetal bovine serum are added and cultured in an incubator at 37° C. Preferably, after the printed material is cultured in DMEM culture medium and 10% fetal bovine serum in an incubator at 37° C., it is transferred to the greater omentum for further incubation.

[0031] Beneficial effects of the present invention:

[0032] The present invention prepares GelMA and Laponite into a GelMA-Laponite composite hydrogel material, which is used as biological ink for 3D printing and has the performance of promoting vascularization.

[0033] The present invention mixes GelMA-Laponite composite hydrogel and seed cells, and uses a 3D bioprinting machine to print a GelMA-Laponite scaffold with a three-dimensional spatial structure on a bladder decellularized matrix. The scaffold is cross-linked using ultraviolet light to obtain a 3D bioprinted double-layer vascularized bladder patch. The present invention provides a bladder patch, which uses GelMA-Laponite composite hydrogel as a 3D printing material and a bladder decellularized matrix as a substrate. Adipose stem cells are encapsulated inside the 3D printing material. Adipose stem cells can effectively differentiate toward vascular endothelial cells using magnesium ions and silicon ions in Laponite, giving the bladder patch the function of promoting the formation of a vascular network. In addition, by adjusting the 3D bioprinting parameters to control the spatial positioning of seed cells and materials, a complex three-dimensional tissue structure is constructed, which is conducive to the proliferation of adipose stem cells in the scaffold and improves the repair effect of the patch. At the same time, the pores in the scaffold reserve space for the vascular endothelial cells in the surrounding tissue to migrate and proliferate to the patch, thereby realizing the construction of a tissue engineering bladder patch with a complex vascular regulatory network structure. Through omentum encapsulation and incubation, the maturation and directional regulation of the patch vascular network system are further promoted. The above design promotes the vascularization performance of the bladder patch through three levels. The present invention is expected to improve the construction level of tissue-engineered bladder patches, solve the vascularization problem of tissue-engineered bladder transplants, and lay a good theoretical foundation and technical support for promoting the clinical application of bladder tissue engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 This is the preparation result of the GelMA hydrogel in Example 1, wherein:

[0036] A: shows the pure GelMA obtained after lyophilization;

[0037] B: shows the Fourier transform infrared spectroscopy detection results of GelMA hydrogel.

[0038] Figure 2Comparison chart of fluidity of hydrogels with different concentrations, including: A, 10% GelMA; B, 0.5% GelMA-Laponite; C, 1% GelMA-Laponite; D: 2% GelMA-Laponite.

[0039] Figure 3 The swelling curves and degradation curves of composite hydrogels with different concentrations are shown;

[0040] A: Swelling curves of composite hydrogels with different concentrations;

[0041] B: Degradation curves of composite hydrogels with different concentrations.

[0042] Figure 4 Shows the compression properties of composite hydrogels with different concentrations; among them,

[0043] A: Compression performance results of composite hydrogels with different concentrations;

[0044] B: Cyclic compression test results of composite hydrogels with different concentrations.

[0045] Figure 5 The rheological properties of composite hydrogels with different concentrations are shown, among which,

[0046] A: Viscosity-shear rate curve;

[0047] B: Temperature-viscosity curve.

[0048] Figure 6 Shows the biocompatibility results of Laponite hydrogel extracts with different concentrations.

[0049] Figure 7 Shows the biocompatibility test results of Laponite hydrogels with different concentrations under three-dimensional culture.

[0050] Figure 8 Show the 3D printing performance of composite hydrogel bio-ink; among them,

[0051] A: The bio-ink is extruded in filaments during the 3D printing process;

[0052] B: 3D printing mesh support process;

[0053] C: 3D printed mesh finished product;

[0054] D, E: The conditions of cells in the scaffold on the 1st day and the 7th day were observed under a microscope, and the proliferation effect of cells in the scaffold was obvious;

[0055] F, G: 3D printed “301” text and hollow cylindrical structure.

[0056] Fig. 9 Showing the live-dead staining results of cells cultured in the printed scaffold on the seventh day.

[0057] Fig.10 The following are photos of bladder patches, including: A: bladder after washing; B: bladder decellularized matrix after freeze-drying; C: round bladder decellularized matrix patch with a diameter of 6 mm; D: double-layer bladder patch.

[0058] Fig.11 Shows the results of the scratch test and tube formation test.

[0059] Fig.12 The qPCR results show the expression levels of different vascular endothelial cell-related genes in the adipose stem cells of the 10% GelMA group and the 1% GL group after induction of differentiation. DETAILED DESCRIPTION

[0060] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0061] Laponite XLG(Mg 5.34 Li 0.66 Si8O 20 (OH)4Na 0.66 , Clay), whose Chinese name is lithium magnesium silicate or nano clay, is provided by Rockwood Company of the United States.

[0062] The bio-ink of the present invention is a composite hydrogel solution made of methacrylic anhydride gelatin solution and Laponite aqueous dispersion, wherein the concentration of methacrylic anhydride gelatin is 5-15% (w / v), and the concentration of Laponite is 0.2-2% (w / v).

[0063] Preferably, the methacrylic anhydride gelatin solution is obtained by dissolving freeze-dried methacrylic anhydride gelatin in a buffer solution at 37°C.

[0064] The buffer solution includes one or more of phosphate buffer solution, Tris buffer solution and borate buffer solution.

[0065] Preferably, the buffer solution is phosphate buffer solution (PBS, pH=7.4).

[0066] The methacrylic anhydride gelatin solution also contains a photoinitiator curing agent, which includes one or two of phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone. The concentration of the photoinitiator is 0.2-0.5% (w / v), preferably, the concentration of the photoinitiator is 0.25% (w / v).

[0067] Phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP photoinitiator), LAP photoinitiator is a blue light initiator. Under the action of blue light (wavelength 405nm), LAP quickly initiates the curing of GelMA material.

[0068] Methacrylic anhydride gelatin can be obtained by a common method in the art. For example, an embodiment of the present invention provides an optional method for preparing methacrylic anhydride gelatin, comprising the following steps:

[0069] Take gelatin and Na2CO3, add them to ultrapure water, and prepare a homogeneous solution with a concentration of about 10% (w / v). Heat and stir to fully dissolve the gelatin until it presents a uniform light yellow color. Then, add methacrylic anhydride to the gelatin solution. After the addition is completed, let the mixed solution react at 50°C for 3 hours, and adjust the pH of the mixed solution to 9. After the reaction is completed, add PBS buffer to dilute and terminate the reaction.

[0070] The mixed solution after the reaction is poured into a dialysis bag to remove salts, unreacted methacrylic anhydride and other by-products. The mixture is filtered and centrifuged to obtain the supernatant to obtain methacrylic anhydride gelatin.

[0071] Methacrylic anhydride gelatin was freeze-dried for 5 days using a freeze dryer to obtain GelMA material.

[0072] The embodiment of the present invention includes a method for preparing a biological ink, wherein a methacrylic anhydride gelatin solution and a Laponite dispersion are taken and stirred to obtain a composite hydrogel solution, namely the biological ink (GelMA-Laponite composite hydrogel). Specifically,

[0073] Disperse ultraviolet sterilized Laponite XLG in deionized water and stir vigorously to obtain a clear Laponite dispersion; preferably, the concentration of the Laponite dispersion is 0.4-4% (w / v), or 1.5-2.5% (w / v), that is, the concentration of the Laponite dispersion can be any value in the above range, for example, 0.4%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5% or 4%, etc.

[0074] Take GelMA material, dissolve it in PBS buffer at 37°C, add LAP photoinitiator to prepare methacrylic anhydride gelatin solution (GelMA solution), preferably, the concentration of GelMA solution is 10-30% (w / v), or 15-25% (w / v), that is, the concentration of GelMA solution can be any value within the above range, for example, 10%, 15%, 20%, 25% or 30%, etc.

[0075] The Laponite dispersion and the filtered and sterilized GelMA solution were mixed in equal volumes and stirred to obtain the bio-ink.

[0076] An embodiment of the present invention also provides a method for preparing a 3D bioprinted bladder patch, comprising the following steps:

[0077] The bladder decellularized matrix was loaded into a cell culture dish, wherein the muscle layer of the bladder decellularized matrix faced upward, and the culture dish was placed on the cooling platform of the 3D bioprinter.

[0078] The cell-laden composite hydrogel bio-ink was loaded into a sterile syringe, the printing needle was replaced, and 3D printing was performed according to the following parameters: the syringe temperature was set to 25°C, the platform temperature was 4°C, the printing speed was 2mm / s, and the extrusion rate was 0.3mm 3 / s, the printing needle is G21 needle.

[0079] Adipose-derived stem cells (ADSCs) were selected, and the p3-p6 cells were digested with trypsin and centrifuged. The supernatant was aspirated and evenly resuspended with a certain amount of composite hydrogels of different concentrations to obtain composite hydrogels loaded with cells, wherein the concentration of adipose-derived stem cells was 1x107 / mL.

[0080] After printing, the printed material was cross-linked under 405 nm ultraviolet light for 40 seconds, and then DMEM culture medium and 10% fetal bovine serum were added and cultured in a 37°C incubator. The printed material was transferred to the greater omentum for further incubation to obtain a double-layer bladder patch.

[0081] Example 1

[0082] 1. Preparation of GelMA

[0083] 10g of type A gelatin from pig skin and 5g of Na2CO3 were weighed on a precision electronic analytical balance and added to 100mL of ultrapure water to prepare a 10% (w / v) homogeneous solution. The solution was transferred to a heated magnetic stirrer and stirred at 250rpm in a 50°C water bath to fully dissolve the gelatin until a uniform light yellow gelatin solution was obtained.

[0084] Add 1 mL of methacrylic anhydride dropwise to the gelatin solution (the addition time is controlled within about 2 minutes). After the addition is completed, cover the reaction container with tin foil and allow the mixed solution to react at 50°C for 3 hours. Adjust the pH to 9 with 10% (w / v) NaOH solution every 30 minutes. After the reaction is completed, add PBS buffer to dilute and terminate the reaction.

[0085] The mixed solution after the reaction was poured into a dialysis bag and dialyzed with deionized water at 50° C. for about 7 days (changing the water 3 times a day) to remove salts, unreacted methacrylic anhydride and other by-products.

[0086] Afterwards, after filtering with a 0.22 μm filter membrane, the mixed solution was transferred to a centrifuge tube, centrifuged at 6000 rpm for 5 min, and the supernatant was placed in a 50 ml test tube, frozen overnight in a -80 °C ultra-low temperature refrigerator, and then freeze-dried for 5 days using a freeze dryer. The obtained dry white foam sample, i.e., GelMA hydrogel, was stored in a -20 °C low temperature refrigerator for further use.

[0087] like Figure 1 A shows the pure GelMA material obtained after freeze-drying, which has a white loose foam-like structure; Figure 1 B shows the Fourier transform infrared spectrum test results of GelMA, and the control group is gelatin material. The stretching vibration of C=O bond, bending vibration of NH bond, and in-plane bending vibration of CN bond and NH bond in the chemical structure lead to the appearance of 1628cm -1 (amide I), 1538cm -1 (amide II) and 1480 cm -1 The characteristic peak of (amide III) indicates that GelMA modification is successful and double bonds have been introduced.

[0088] 2. Synthesis of GelMA-Laponite Composite Hydrogel

[0089] UV sterilized Laponite XLG was dispersed in deionized water and stirred at 300 rpm with a magnetic stirrer for 30 minutes to obtain a clear 2% (w / v) Laponite dispersion.

[0090] The synthesized GelMA hydrogel was dissolved in PBS (pH=7.4) at 37°C, 0.25% (w / v) LAP photoinitiator was added to the solution to prepare a 20% (w / v) GelMA solution, and the GelMA solution was filtered and sterilized using a 0.22 um filter.

[0091] 20% (w / v) GelMA solution and 2% (w / v) Laponite dispersion were mixed in equal volume ratios and vigorously stirred for 4 hours to obtain a GelMA-Laponite composite hydrogel solution, wherein the concentration of GelMA was 10% (w / v) and the concentration of Laponite was 1% (w / v).

[0092] 3. Preparation of Bladder Decellularized Matrix

[0093] Complete rat bladder tissue was obtained. The mucosal layer was washed three times in PBS buffer, then frozen at -80°C for at least 24 hours. After thawing at room temperature, surgical scissors and forceps were used to separate and remove the muscle layer and serosal layer of the bladder under a microscope, leaving the complete mucosal layer. The complete mucosal layer tissue was placed in a plastic box, 1% Triton X-100 was added and soaked on a shaker (60r / min) for 6 hours for decellularization, and then treated with deionized water for 2 hours. Then 1% sodium dodecyl sulfate (SDS) was added and soaked on a shaker (60r / min) for 2 hours, and finally soaked in deionized water for 1 hour to wash away the residual decellularization solution, and then freeze-dried. During the bladder decellularization process, the bladder tissue gradually changed from pink to white at the beginning, and the bladder decellularized matrix was obtained. The freeze-dried decellularized matrix was cut into circular patches with a diameter of 6 mm with scissors. All circular patches were sterilized by Co 60 irradiation and stored for subsequent preparation of double-layer patches.

[0094] 4. Preparation of 3D Bioprinted Vascularized Bladder Patch

[0095] The bladder printing data model was constructed using 3D MAX software and exported as an STL file. The 3D bioprinter (SunP BioMaker 4) was started, the inside of the 3D bioprinter was wiped with gauze containing 75% alcohol, and sterilized by ultraviolet irradiation for 40 minutes.

[0096] A 6mm diameter round bladder decellularized matrix was loaded into a 60mm cell culture dish, with the muscle layer facing upwards, and the dish was placed on the cooling platform of the 3D bioprinter. A pre-designed 3D data model of a tissue engineering bladder patch that simulates the anatomical structure of the bladder wall was found.

[0097] The syringe temperature was set to 25°C, the platform temperature to 4°C, the printing speed to 2mm / s, and the extrusion rate to 0.3mm. 3 / s, the printing needle is G21 needle.

[0098] Adipose-derived stem cells (ADSCs) were selected, and the p3-p6 cells were digested with trypsin and centrifuged. The supernatant was aspirated and evenly resuspended with a certain amount of composite hydrogels of different concentrations to obtain composite hydrogels loaded with cells, where the concentration of adipose-derived stem cells was 1x10 7 / mL.

[0099] Load the cell-laden composite hydrogel bio-ink into a 5 ml sterile syringe and replace it with a sterile printing needle.

[0100] After printing, the printed material was exposed to 405nm ultraviolet light at 80mw / cm 2 The printed material was cross-linked with light intensity of 40 seconds, and then DMEM medium and 10% fetal bovine serum were added and cultured in an incubator at 37°C. After two days of culture, the printed material was transferred to the greater omentum and incubated at 37°C for another two weeks.

[0101] Example 2 Preparation of composite hydrogels of different concentrations

[0102] UV sterilized Laponite XLG was dispersed in deionized water and stirred at 300 rpm with a magnetic stirrer for 30 minutes to obtain clear 1% (w / v), 2% (w / v) and 4% (w / v) Laponite dispersions.

[0103] The synthesized GelMA hydrogel was dissolved in PBS (pH=7.4) at 37°C, 0.25% (w / v) LAP photoinitiator was added to the solution to prepare a 20% (w / v) GelMA solution, and the GelMA solution was filtered and sterilized using a 0.22 um filter.

[0104] 20% (w / v) GelMA solution and 1% (w / v), 2% (w / v), and 4% (w / v) Laponite dispersions were mixed in equal volume ratios and vigorously stirred for 4 hours to obtain a composite hydrogel solution with a final concentration of 10% w / v GelMA and 0.5% (w / v), 1% (w / v), and 2% (w / v) Laponite.

[0105] The results are as follows Figure 2 As shown, with the increase of Laponite concentration, the fluidity of the composite hydrogel becomes worse and worse, and the color gradually changes from transparent to turbid, among which the 2% GL composite hydrogel has become a solid gel with poor fluidity.

[0106] Example 3 Swelling experiment and degradation experiment

[0107] Swelling test:

[0108] 200uL of the composite hydrogels of the four concentrations prepared in Example 2 were respectively taken into a homemade cylindrical mold, cross-linked by 405nm UV light for 40s, placed in a -20°C refrigerator overnight, and freeze-dried in a freeze dryer for 2-3 days. Three samples were prepared for each of the above concentrations.

[0109] The weight of the freeze-dried sample was weighed and recorded as the initial weight W0. The sample was placed in a 24-well plate and 1 mL of PBS was added. The water on the surface of the sample was gently wiped with absorbent paper and the weight was weighed after 1, 3, 6, 12, 24, 36, and 48 hours, respectively, and recorded as Wt.

[0110] The swelling ratio at each time point is (Wt-W0) / W0×100%.

[0111] Degradation experiment:

[0112] The previous steps were the same as the swelling experiment. The weight measured after 48 hours of swelling equilibrium was taken as the initial weight W0. Collagenase was added and the weight was measured at 2, 4, 6, and 8 hours respectively as Wt.

[0113] The degradation rate at different time points is (W0-Wt) / W0×100%.

[0114] like Figure 3 As shown in A, the swelling properties of the composite hydrogel vary with the change of Laponite concentration. The interaction force between Laponite and GelMA inhibits the swelling properties of the GL composite hydrogel. The swelling properties of the 10% GelMA group are the highest, and the swelling properties of the 2% GL composite hydrogel group are the lowest. This reduction in swelling is beneficial for the printed scaffold to maintain its original shape in a humid environment.

[0115] like Figure 3 As shown in Figure B, all hydrogels showed biodegradation over time. However, under the same experimental conditions, the degradation rates of hydrogels with different components were different, among which 10% GelMA degraded the fastest, while 2% GL composite hydrogel degraded the slowest.

[0116] Example 4 Investigation of the compression capacity of the composite hydrogel

[0117] 200uL of the four concentrations of composite hydrogel prepared in Example 2 were respectively taken into a homemade mold and cross-linked by 405nm UV light for 40s. Each group of hydrogels was cross-linked into a cylinder (height 5mm, diameter 10mm), with 3 samples for each concentration. The compression properties were then tested using a universal mechanical detector, and the compression strain rate was 1mm / min.

[0118] like Figure 4 As shown in A, the compression properties of the composite hydrogels vary with the concentration of Laponite, and the compression properties of the 2% composite hydrogel group are the best.

[0119] like Figure 4 As shown in B, composite hydrogels with different concentrations were subjected to cyclic compression tests. The results are consistent with the simple compression performance test, and show that composite hydrogels of each concentration have good fatigue resistance.

[0120] Example 5 Investigation of the rheological properties of the composite hydrogel

[0121] Using a rheological property tester, 150uL of hydrogel was placed on the test bench, and the different rheological properties (such as shear-viscosity, temperature-viscosity, etc.) of the four concentrations of hydrogel liquid prepared in Example 2 were tested respectively, and each concentration and each test were measured three times.

[0122] like Figure 5 As shown in the viscosity-shear rate curve of A, the viscosity of the composite hydrogel changes with the change of Laponite concentration, among which the initial viscosity of the 2% GL composite hydrogel group is the highest. However, the viscosity is almost the same at high speed, indicating that the composite hydrogel has good shear thinning properties and is more suitable for extrusion bioprinting.

[0123] like Figure 5 As shown in the temperature-viscosity curve of B, the temperature-viscosity curve of the composite hydrogel gelation changes with the change of Laponite concentration. The results show that this change broadens the temperature window of 3D printing, and the 2% GL composite hydrogel enables 3D printing to proceed normally at room temperature.

[0124] Example 6 Investigation of the biocompatibility of the composite hydrogel

[0125] Take four culture dishes, add 1 mL of the four concentrations of composite hydrogel prepared in Example 2 respectively, add 10 mL of complete culture medium after UV curing, incubate at 37°C for one day, aspirate the culture medium, filter and sterilize, and then use it to culture adipose stem cells, add live-dead dye at different culture time points, and observe the cell activity under different material extract culture under a microscope.

[0126] like Figure 6 As shown, in the biocompatibility test of Laponite hydrogel extracts with different concentrations, the 1% GL composite hydrogel group had better biocompatibility and higher cell viability.

[0127] Example 7 Investigation of the biocompatibility of composite hydrogels in three-dimensional culture

[0128] Adipose stem cells were mixed with the four concentrations of hydrogels prepared in Example 2, respectively, and then dropped into a well plate. After photo-crosslinking, complete culture medium was added for culture. At different time points, CCK dye was added to the well plate, and cell activity was detected by a microplate reader.

[0129] like Figure 7 As shown, it can be seen that the 1% GL composite hydrogel group has the best biocompatibility in three-dimensional culture, while the biocompatibility of the 10% GelMA group and the 2% GL composite hydrogel group is relatively poor.

[0130] In addition, the above examples prove that the 1% GL composite hydrogel has excellent compression properties, which is sufficient to withstand the continuous deformation of the bladder during urine storage and urination. The good rheological properties enable it to complete the 3D printing process more perfectly than lower concentration bio-inks, and it will not have a viscosity similar to higher concentration bio-inks to cause clogging of the printing needle. Compared with lower concentration bio-inks, it has lower swelling properties, so it will not swell excessively due to the humid environment of the bladder, and the longer degradation time enables it to ensure perfect tissue repair. This moderate swelling and degradation performance makes it very suitable for subsequent bladder patch applications. Therefore, it was decided to use 1% concentration GL composite hydrogel for subsequent experiments.

[0131] Example 8 3D printing of hydrogel bio-ink

[0132] 1% concentration of GL composite hydrogel was used for 3D printing, and the printing process was the same as in Example 1.

[0133] A: GelMA-Laponite hydrogel bio-ink is extruded in a 3D printer in a filamentous shape, with a diameter close to that of a needle; B, C: 3D printing mesh scaffold process and finished product pictures. D, E: The condition of cells in the scaffold on the 1st and 7th days was observed under a microscope, and the cell proliferation effect in the scaffold was obvious. In the three-dimensional grid, the junction of two vertical filaments shows good interconnectivity between adjacent layers, and a clear composite spatial structure can be observed.

[0134] F, G: 3D printed the "301" text and hollow cylindrical structure, and optimized the 3D printing parameters.

[0135] During the 3D printing process, if the syringe temperature is higher than 25°C, the printing speed is lower than 1.5mm / s, or the extrusion rate is greater than 0.35mm 3 / s, the bio-ink extrudate is droplet-shaped rather than filamentous, the printed material is irregular and adjacent printed lines merge with each other, making the scaffold pores smaller or disappear. On the contrary, if the syringe temperature is lower than 25°C, the printing speed is greater than 1.5mm / s or the extrusion rate is less than 0.35mm 3 / s, there will be problems such as blockage of the printing syringe, viscous and irregular extrudates, discontinuous print lines, etc., and the composite spatial structure of the printed object cannot be formed. The above two situations will lead to the failure of the spatial structure construction of 3D bioprinting, resulting in the inability of seed cells to accurately locate in the material 3D scaffold and affecting the migration of surrounding vascular endothelial cells.

[0136] Example 9 Cell live and dead staining experiment

[0137] The cell-laden bio-ink was stained with live-dead stain on the 7th day after being printed into a scaffold, and the cell activity in the scaffold was photographed using a confocal microscope.

[0138] like Fig. 9 As shown in b, different scaffolds loaded with cells were cultured in vitro for 7 days, and then the cells were stained for live and dead cells. The staining results showed that the live cell density in the 1% GL scaffold group was higher than that in the 10% GelMA group, indicating that the 1% GL scaffold environment was more suitable for cell growth and proliferation.

[0139] Fig.10 A: Washed bladder; B: Freeze-dried bladder decellularized matrix. C: 6mm diameter round bladder decellularized matrix patch. D: Double-layer bladder patch, the upper layer is 3D printed scaffold, the lower layer is bladder decellularized matrix disc.

[0140] Example 10 Scratch test and tube forming test

[0141] Tube forming experiment:

[0142] Before the experiment, the well plate and the pipette tip were precooled in a 4℃ refrigerator, and the matrix gel was melted at 4℃. Take a 24-well plate, add 20-30uL matrix gel to each well, and spread it evenly on the wall but do not let the gel touch the wall of the well. After laying, put it in a 37℃ incubator for 30-60min to solidify the matrix gel. Add cells, 150,000-200,000 cells per well, and add ECM complete culture medium, 10% GelMA ECM extract, and 1% GL ECM extract respectively. A group of three duplicate wells were incubated at 37℃ for 12h, and then the live-dead stain was added to take pictures of the tube effect under a microscope. The pictures were analyzed with imageJ software.

[0143] Scratch test:

[0144] Plant 200,000 to 300,000 cells in each well of a 6-well plate. When the cells proliferate to more than 90%, use a 200uL pipette tip to draw a vertical line in the center of the well plate. Aspirate the culture medium, add PBS for washing, and then replace with serum-free or low-serum culture medium. Photograph the scratch area at 0h under a microscope, put it in an incubator for culture, take it out after 24h, and photograph the scratch area at 24h. Including control group (ECM culture medium culture), 10% GelMA group (ECM material extract) and 1% GL group (ECM material extract). After shooting, use imageJ software to analyze the migration area.

[0145] like Fig.11 As shown, compared with the control group and the 10% GelMA group, the cell migration area of ​​the 1% GL group was the largest, about 52%. The tube formation result of the 1% GL group was also the best, with about 188 nodes formed and a total length of about 15001px.

[0146] From the results, it can be seen that 1% GL composite hydrogel has an extremely superior promoting effect on the migration and tube formation of vascular endothelial cells.

[0147] Example 11 qPCR experiment

[0148] 1. RNA extraction: The experiment was divided into a control group, a 10% GelMA group and a 1% GL group. The control group was a two-dimensional induction culture, and the other two groups were a three-dimensional induction culture. The induction medium was an ECM complete medium. The detection time points for each group were 4 days, 7 days and 14 days. At the detection time point, the culture medium of each group was aspirated, washed with PBS and then aspirated, and the prepared 0.3mg / mL GelMA lysate was taken, 500uL was added to each well, and placed in a 37℃ incubator for 1 hour, and the state was observed under a microscope every 10 minutes. After each group of hydrogels was completely lysed, the liquid in the well was aspirated into the EP tube and centrifuged at 1000rpm for 5min. After taking it out, the supernatant was removed, PBS was added for washing and centrifuged to remove the supernatant. The precipitate was resuspended with 1mL Trizol, 200uL chloroform was added and shaken evenly. Centrifuged at 12000rpm for 15min at 4℃, 400uL of the upper supernatant was taken into a new EP tube, an equal volume of isopropanol was added, and centrifuged at 12000rpm for 10min after shaking. Discard the supernatant, add 1 mL of 75% ethanol solution and centrifuge at 12000 rpm for 5 min. Aspirate the ethanol and dry the residual ethanol, add 20 uL of enzyme-free water and measure the RNA concentration.

[0149] 2. RNA reverse transcription: Configure the standard reverse transcription 20u system and then perform reverse transcription in the machine.

[0150] 3. PCR: Also prepare 20uL of common PCR system, where primers are GAPDH, PECAM1, ANGPT1 and KDR (GAPDFH is the internal reference gene and the control group is the negative control). Put it into the machine for PCR experiment, and finally export the results for calculation.

[0151] like Fig.12 As shown in the figure, qPCR experiments showed that compared with the 10% GelMA group, the expression levels of different endothelial cell-related genes in the 1% GL composite hydrogel group after induced differentiation were relatively higher, and the gene expression level was the highest on the 7th day of culture. This phenomenon proves that the 1% GL composite hydrogel group has a better promoting effect on the differentiation of adipose stem cells into vascular endothelial cells, and the best time is 7 days of culture.

[0152] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A biological ink, characterized in that: The bio-ink is a composite hydrogel solution made of methacrylic anhydride gelatin solution and Laponite dispersion, wherein: The concentration of methacrylic anhydride gelatin is 5 to 15% (w / v). The concentration of Laponite is 0.2 to 2% (w / v).

2. The bio-ink according to claim 1, characterized in that: The methacrylic anhydride gelatin solution is obtained by dissolving freeze-dried methacrylic anhydride gelatin in a buffer solution.

3. The biological ink according to claim 2, characterized in that: The buffer solution includes one or more of a phosphate buffer solution, a Tris buffer solution, and a borate buffer solution.

4. The method for preparing the biological ink according to claim 1, characterized in that: The preparation method comprises the following steps: The methacrylic anhydride gelatin solution is mixed with the Laponite dispersion and stirred to obtain a composite hydrogel solution, which is the bio-ink.

5. A 3D printed biological bladder patch, characterized in that: The bladder patch comprises a bladder decellularized matrix and a 3D printed scaffold, wherein the 3D printed scaffold is 3D printed on the bladder decellularized matrix by the biological ink described in claim 1.

6. The method for preparing the 3D printed biological bladder patch according to claim 5, characterized in that: The preparation method comprises the following steps: (1) placing the bladder decellularized matrix in a 3D bioprinter with the muscle layer of the bladder decellularized matrix facing upward; (2) 3D printing of cell-laden bio-ink onto a bladder decellularized matrix; (3) After printing is completed, the printed material is cross-linked with ultraviolet light and incubated.

7. The method for preparing the biological bladder patch according to claim 6, characterized in that: In step (2), the 3D printing parameters are that the syringe temperature is set to 20-25°C, and / or, The platform temperature is 3-6°C, and / or, Printing speed is 1.5-2.5mm / s, and / or, Extrusion rate is 0.25-0.35mm 3 / s.

8. The method for preparing the biological bladder patch according to claim 6, characterized in that: In step (3), the printed material is irradiated with 405 nm ultraviolet light for 40 seconds at an intensity of 80 mW / cm 2 .

9. The method for preparing the biological bladder patch according to claim 6, characterized in that: In step (3), after the printed material is cross-linked by ultraviolet light, DMEM culture medium and 10% fetal bovine serum are added and cultured in an incubator at 37°C.

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