Bladder acellular matrix hydrogel, 3D bio-printing bladder patch as well as preparation method and application of bladder acellular matrix hydrogel and 3D bio-printing bladder patch

Abstract

The invention belongs to the technical field of 3D biological printing, and particularly relates to bladder acellular matrix hydrogel, a 3D biological printing bladder patch and a preparation method and application of the bladder acellular matrix hydrogel and the 3D biological printing bladder patch. The bladder acellular matrix hydrogel is prepared from a methacrylated bladder acellular matrix and a photoinitiator. The bladder acellular matrix is firstly subjected to methacrylation, so that the bladder acellular matrix has the photo-crosslinking gelling characteristic, the rheological property of the bladder acellular matrix hydrogel can be improved, the mechanical stability of the bladder acellular matrix hydrogel can be enhanced, and meanwhile, the bladder acellular matrix hydrogel has the environmental characteristic of simulating a bladder tissue extracellular matrix and does not cause immune response. Bio-ink prepared from the bladder acellular matrix hydrogel has the characteristics of shear thinning and temperature response capability, and bladder tissue engineering patches can be printed by using a 3D bio-printer.

Description

Technical Field

[0001] The present invention belongs to the technical field of 3D bioprinting, and specifically relates to a bladder acellular matrix hydrogel, a 3D bioprinted bladder patch, and a preparation method and application thereof. Background Art

[0002] Congenital and acquired bladder diseases such as bladder exstrophy, neurogenic bladder, malignant tumors and trauma can lead to bladder defects and dysfunction, which require surgical repair to protect bladder and kidney function. In severe cases, radical cystectomy and urinary diversion are required, which will have a serious impact on the patient's quality of life.

[0003] At present, the use of intestinal tubes for repair and reconstruction after radical cystectomy is a conventional treatment for bladder function reconstruction in clinical practice. However, due to the obvious differences in tissue structure and function between the intestine and the bladder, the use of intestinal tubes to repair and reconstruct the bladder is often accompanied by a series of complications, such as mucus secretion, long-term bacteriuria, stone formation and fibrosis. The emergence and development of tissue engineering technology has provided new ideas for the repair and reconstruction of the bladder.

[0004] The selection of scaffold materials is particularly important in tissue engineering research. Scaffold materials provide support for tissue repair or regeneration. At the same time, they simulate the extracellular matrix environment of regenerated tissue and provide a suitable "microenvironment" for the adhesion, growth, proliferation and differentiation of seed cells.

[0005] Decellularized matrix materials in natural materials remove cellular components that can cause immune responses and retain the extracellular matrix structure and components of the organ or tissue itself, which is conducive to cell adhesion, growth, proliferation and differentiation, and is widely used in bladder tissue engineering research. Previous studies have reported that the prepared bladder decellularized matrix well retains a variety of growth factors, such as vascular endothelial growth factor, platelet-derived growth factor-BB and keratinocyte growth factor. When the bladder decellularized matrix is ​​digested, dissolved and polymerized by pepsin to form a hydrogel, the growth factors in the bladder decellularized matrix are still retained.

[0006] 3D bioprinters can precisely control the positioning of functional components such as biomaterials, biochemical agents and living cells in different layers by printing layer by layer, thereby simulating the normal physiological structure of the target organ. It is currently a research hotspot in the field of tissue engineering organ regeneration and has been initially applied to tissue engineering regeneration of tissues and organs such as skin, vascular system and cartilage.

[0007] In addition to having good rheological properties that are beneficial to 3D printing, "bio-ink" used in 3D bioprinting should also have properties that support cell growth, proliferation, and tissue formation. Among them, hydrogel is the main component of "bio-ink" required for 3D bioprinting. How to use the decellularized matrix of tissues or organs themselves to prepare 3D bioprinting hydrogels with good physical and chemical properties has received extensive attention and research.

[0008] Hydrogels prepared from decellularized matrices have attracted much attention in the field of tissue engineering because of their ability to simulate the extracellular matrix environment of tissues or organs and their good biocompatibility. However, due to their poor mechanical stability, they are often used as cell carriers for constructing engineered tissues, and are difficult to use alone as bioprinting inks for 3D bioprinting. In the study of 3D bioprinting of bladder tissue engineering, there have been no reports on the use of bladder decellularized matrix hydrogels as bioinks for 3D bioprinting. Summary of the invention

[0009] Based on the above technical background, the main purpose of the present invention is to provide a bladder decellularized matrix hydrogel, a 3D bioprinted bladder patch and a preparation method and application thereof, so as to overcome the deficiencies in the prior art.

[0010] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention includes:

[0011] The first aspect of the present invention is to provide a bladder decellularized matrix hydrogel, wherein the bladder decellularized matrix hydrogel is prepared by methacrylylated bladder decellularized matrix and a photoinitiator;

[0012] The mass ratio of the methacrylylated bladder decellularized matrix to the photoinitiator is 1:(0.2-0.3).

[0013] Preferably, the methacrylated bladder decellularized matrix is ​​prepared from bladder decellularized matrix and methacrylic anhydride;

[0014] The mass ratio of the bladder decellularized matrix to methacrylic anhydride is 1:(0.5-0.7).

[0015] Preferably, the photoinitiator is selected from one or more of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), Irgacure 2959, and 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone.

[0016] The second aspect of the present invention is to provide a method for preparing the bladder decellularized matrix hydrogel according to the first aspect of the present invention, the preparation method comprising the following steps:

[0017] The photoinitiator is dissolved in a solvent to obtain a photoinitiator solution, the methacrylylated bladder decellularized matrix is ​​added to the photoinitiator solution to obtain a photo-crosslinked bladder decellularized matrix hydrogel precursor solution, and then the solution is placed under light conditions for crosslinking reaction to obtain a bladder decellularized matrix hydrogel.

[0018] Preferably, the method for preparing the methacrylylated bladder acellular matrix comprises the following steps:

[0019] The bladder decellularized matrix is ​​freeze-dried to obtain freeze-dried powder of the bladder decellularized matrix. The freeze-dried powder of the bladder decellularized matrix is ​​added to a hydrochloric acid pepsin solution for digestion, and then an alkaline solution is added to adjust the pH. After cooling, methacrylic anhydride is added, and the reaction is stirred under light-proof conditions. Finally, the dialysis is performed with a dialysis membrane and freeze-dried to obtain a methacrylylated bladder decellularized matrix.

[0020] More preferably, the method for preparing the bladder decellularized matrix comprises the following steps:

[0021] The bladder tissue frozen at -80°C was thawed at room temperature, part of the tissue at the top of the bladder was removed and connected to the perfusion decellularization system, the bilateral ureters were used as the inflow channel of the perfusion solution, and the notch at the top of the bladder was used as the outflow channel of the perfusion solution. Triton X-100 with a mass fraction of 0.5-2% was used as the perfusion solution for 4-7 hours, then deionized water was used for perfusion for 0.5-2 hours, and then SDS with a mass fraction of 0.5-2% was used for perfusion for 0.5-2 hours. Finally, it was rinsed with deionized water for 20-30 hours to completely remove the chemical detergent to obtain the bladder decellularized matrix.

[0022] The third aspect of the present invention is to provide a method for preparing a 3D bioprinted bladder patch, the preparation method comprising the following steps:

[0023] Step 1, adding gelatin to the bladder acellular matrix hydrogel described in the first aspect of the present invention, mixing and dissolving, and obtaining 3D printing bio-ink;

[0024] Step 2: Add the 3D printing bio-ink into the 3D bio-printer and perform 3D printing to obtain a 3D bio-printed bladder patch.

[0025] In step 1,

[0026] Preferably, gelatin is added to the bladder decellularized matrix hydrogel, the mass ratio of the bladder decellularized matrix hydrogel to gelatin is 10:(0.4-0.7), and it is dissolved at 35-45° C. to obtain 3D printing bio-ink.

[0027] The fourth aspect of the present invention is to provide a 3D bioprinted bladder patch prepared according to the preparation method of the 3D bioprinted bladder patch described in the third aspect of the present invention.

[0028] The fifth aspect of the present invention is to provide an application of the 3D bioprinted bladder patch described in the fourth aspect of the present invention, and the 3D bioprinted bladder patch can be used as a biological scaffold.

[0029] The beneficial effects of the present invention are:

[0030] (1) The bladder decellularized matrix hydrogel described in the present invention is prepared from methacrylated bladder decellularized matrix and a photoinitiator. The present invention first performs methacrylation on the bladder decellularized matrix to enable it to have photo-crosslinking gelation properties, thereby improving the rheological properties of the photo-crosslinked bladder decellularized matrix hydrogel and enhancing its mechanical stability. At the same time, the bladder decellularized matrix hydrogel has the environmental characteristics of simulating the extracellular matrix of bladder tissue, does not cause an immune response, and can provide an extracellular matrix "microenvironment" for cells.

[0031] (2) The preparation method of the bladder decellularized matrix hydrogel described in the present invention comprises the following steps: first, a self-designed perfusion decellularization system is used to prepare a bladder decellularized matrix. The prepared bladder decellularized matrix not only removes the cellular components that cause immune response, but also well retains the collagen structure and bioactive components, and has the characteristics of supporting cell growth, proliferation and tissue formation; then, the prepared bladder decellularized matrix is ​​modified with methacrylic anhydride to have the characteristics of photo-crosslinking gelation; the bladder decellularized matrix hydrogel can be used to print bladder tissue engineering patches using a 3D bioprinter, thereby establishing a stable and reliable 3D bioprinting system.

[0032] (3) After the bladder decellularized matrix was treated with pepsin digestion, methyl methacrylate anhydride modification, etc., a photo-crosslinked bladder decellularized matrix hydrogel was successfully prepared. Gelatin was added to the bladder decellularized matrix hydrogel to prepare a bio-ink for 3D bioprinting, which has the advantages of both bladder decellularized matrix and gelatin. The bladder decellularized matrix can simulate the microenvironment of bladder tissue, and gelatin gives the bio-ink a good temperature response ability, that is, the bio-ink is in a solid gel state at a lower temperature (such as 4°C) and in a liquid state at a higher temperature (such as 37°C). Rheological characterization shows that the bio-ink has the characteristics of shear thinning and temperature response, and is a good bio-ink for 3D bioprinting. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The photos of bladder tissues before and after decellularization in Example 1 are shown;

[0034] Figure 2 The performance changes of the bladder tissue before and after decellularization in Example 1 are shown:

[0035] Figure B shows the schematic diagram of the change of bladder wall thickness of bladder tissue before and after decellularization;

[0036] Figure C shows the schematic diagram of the changes in DNA content in bladder tissue before and after decellularization;

[0037] Figure D shows the schematic diagram of the changes in collagen content in bladder tissue before and after decellularization;

[0038] Figure E shows the schematic diagram of the changes in GAG content in bladder tissue before and after decellularization;

[0039] Figure 3 A photograph showing the bladder decellularized matrix hydrogel precursor solution prepared in Example 1;

[0040] Figure 4 The photos of the hydrogel state of the bladder decellularized matrix hydrogel prepared in Example 1 before and after light irradiation are shown;

[0041] Figure 5 The viscosity curves of the bladder decellularized matrix hydrogel, gelatin, and bio-ink prepared in Example 1 are shown;

[0042] Figure 6 The temperature scanning curves of the bladder decellularized matrix hydrogel, gelatin and bio-ink prepared in Example 1 are shown;

[0043] Figure 7 Middle: Figure A shows a photo of an extrusion-based 3D bioprinter;

[0044] Figure B shows a photograph of bio-ink extruded into filaments in a 3D bio-printer;

[0045] Figure C shows a photo of the 3D bioprinted grid-shaped multilayer bionic bladder patch (circular patch) prepared in Example 4;

[0046] Figure D shows a photograph of the 3D bioprinted grid-shaped multilayer bionic bladder patch (rectangular patch) prepared in Example 4. DETAILED DESCRIPTION

[0047] The present invention will be described in detail below, and the characteristics and advantages of the present invention will become clearer and more distinct with the description.

[0048] The first aspect of the present invention is to provide a bladder decellularized matrix hydrogel, wherein the bladder decellularized matrix hydrogel is prepared from methacrylylated bladder decellularized matrix and a photoinitiator.

[0049] The mass ratio of the methacrylylated bladder decellularized matrix to the photoinitiator is 1:(0.2-0.3).

[0050] Preferably, the mass ratio of the methacrylylated bladder acellular matrix to the photoinitiator is 1:0.25.

[0051] The methacrylated bladder decellularized matrix is ​​prepared from the bladder decellularized matrix and methacrylic anhydride.

[0052] The mass ratio of the bladder decellularized matrix to methacrylic anhydride is 1:(0.5-0.7).

[0053] Preferably, the mass ratio of the bladder decellularized matrix to methacrylic anhydride is 1:0.6.

[0054] The photoinitiator is selected from one or more of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), Irgacure 2959, and 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone. Preferably, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate.

[0055] The second aspect of the present invention is to provide a method for preparing the bladder decellularized matrix hydrogel according to the first aspect of the present invention, the preparation method comprising the following steps:

[0056] The photoinitiator is dissolved in a solvent to obtain a photoinitiator solution, the methacrylylated bladder decellularized matrix is ​​added to the photoinitiator solution to obtain a photo-crosslinked bladder decellularized matrix hydrogel precursor solution, and then the solution is placed under light conditions for crosslinking reaction to obtain a bladder decellularized matrix hydrogel.

[0057] Preferably, phenyl-2,4,6-trimethylbenzoyl lithium phosphite is dissolved in PBS (phosphate buffered saline) to obtain a photoinitiator solution, wherein the mass concentration of phenyl-2,4,6-trimethylbenzoyl lithium phosphite is 0.2-0.3%, 0.1 g of methacrylated bladder decellularized matrix is ​​added to 5-15 ml of the photoinitiator solution to obtain a photo-cross-linked bladder decellularized matrix hydrogel precursor solution, which is then placed under light of 405 nm wavelength for 20-45 seconds to obtain a bladder decellularized matrix hydrogel.

[0058] More preferably, phenyl-2,4,6-trimethylbenzoyl lithium phosphite is dissolved in PBS (phosphate buffered saline) to obtain a photoinitiator solution, wherein the mass concentration of phenyl-2,4,6-trimethylbenzoyl lithium phosphite is 0.25%, 0.1 g of methacrylated bladder decellularized matrix is ​​added to 10 ml of the photoinitiator solution to obtain a photo-cross-linked bladder decellularized matrix hydrogel precursor solution, which is then placed under light of 405 nm wavelength for 30 seconds to obtain a bladder decellularized matrix hydrogel.

[0059] The method for preparing the methacrylylated bladder decellularized matrix comprises the following steps:

[0060] The bladder decellularized matrix is ​​freeze-dried to obtain freeze-dried powder of the bladder decellularized matrix. The freeze-dried powder of the bladder decellularized matrix is ​​added to a hydrochloric acid pepsin solution for digestion, and then an alkaline solution is added to adjust the pH. After cooling, methacrylic anhydride is added, and the reaction is stirred under light-proof conditions. Finally, the dialysis is performed with a dialysis membrane and freeze-dried to obtain a methacrylylated bladder decellularized matrix.

[0061] Preferably, the freeze-dried bladder decellularized matrix freeze-dried powder is added to a hydrochloric acid pepsin solution with a pH of 1 to 2, and digested at room temperature of 25°C for 45 to 50 hours. The concentration of the hydrochloric acid pepsin solution is 0.5 to 2 mg / ml, and the mass ratio of the bladder decellularized matrix freeze-dried powder to the hydrochloric acid pepsin is 1: (80 to 120). Then, sodium hydroxide solution is added to adjust the pH to 8 to 9. After cooling to 0 to 5°C, methacrylic anhydride is added, and the reaction is stirred for 20 to 30 hours under light-proof conditions. During the reaction, the pH is maintained at 8 to 9. Finally, a 12 to 14 KDa dialysis membrane is used for dialyzing at 0 to 5°C for 1 to 2 days. After freeze-drying, a methacrylylated bladder decellularized matrix is ​​obtained.

[0062] More preferably, the freeze-dried bladder decellularized matrix freeze-dried powder is added to a pepsin hydrochloride solution with a pH of 1, and digested at room temperature of 25°C for 48 hours. The concentration of the pepsin hydrochloride solution is 1 mg / ml, and the mass ratio of the bladder decellularized matrix freeze-dried powder to pepsin hydrochloride is 1:100. Then, sodium hydroxide solution is added to adjust the pH to 8-9. After cooling to 4°C, methacrylic anhydride is added, and the reaction is stirred for 24 hours under light-proof conditions. During the reaction, the pH is maintained at 8-9. Finally, a 12-14KDa dialysis membrane is used for dialyzing at 4°C for 2 days, followed by overnight at -80°C and freeze drying in a freeze dryer for 2-3 days to obtain a methacrylylated bladder decellularized matrix.

[0063] The method for preparing the bladder decellularized matrix comprises the following steps:

[0064] The bladder tissue frozen at -80°C was thawed at room temperature, part of the tissue at the top of the bladder was removed and connected to the perfusion decellularization system, the bilateral ureters were used as the inflow channel of the perfusion solution, and the notch at the top of the bladder was used as the outflow channel of the perfusion solution. Triton X-100 with a mass fraction of 0.5-2% was used as the perfusion solution for perfusion for 4-7 hours, then deionized water was used for perfusion for 0.5-2 hours, and then SDS (detergent) with a mass fraction of 0.5-2% was used for perfusion for 0.5-2 hours. Finally, it was rinsed with deionized water for 20-30 hours to completely remove the chemical detergent to obtain the bladder decellularized matrix.

[0065] Preferably, the bladder tissue frozen at -80°C is thawed at room temperature, and the bladder tissue at the top of the bladder is cut off and connected to the perfusion decellularization system to change the flow direction of the perfusion solution. The bilateral ureters serve as the inflow channel of the perfusion solution, and the notch at the top of the bladder serves as the outflow channel of the perfusion solution. First, 1% Triton X-100 is used as the perfusion solution for 6 hours, then deionized water is used for 1 hour, and then 1% SDS (detergent) is used for 2 hours. Finally, it is rinsed with deionized water for 24 hours to completely remove the chemical detergent to obtain the bladder decellularized matrix.

[0066] The third aspect of the present invention is to provide a method for preparing a 3D bioprinted bladder patch, the preparation method comprising the following steps:

[0067] Step 1, adding gelatin to the bladder acellular matrix hydrogel described in the first aspect of the present invention, mixing and dissolving, and obtaining 3D printing bio-ink;

[0068] Step 2: Add the 3D printing bio-ink into the 3D bio-printer and perform 3D printing to obtain a 3D bio-printed bladder patch.

[0069] The above steps are described in detail below.

[0070] In step 1, gelatin is added to the bladder decellularized matrix hydrogel, the mass ratio of the bladder decellularized matrix hydrogel to gelatin is 10:(0.4-0.7), and it is fully dissolved at 35-45° C. to obtain 3D printing bio-ink.

[0071] Preferably, the mass ratio of bladder decellularized matrix hydrogel to gelatin is 10:0.5, and it is fully dissolved at 40° C. to obtain 3D printing bio-ink.

[0072] In step 2, 3D printing bio-ink was added to the syringe barrel, the air was exhausted, and the syringe was connected to the 3D bio-printer. The printing parameters were: the inner diameter of the printing needle was 0.25 mm, the printing layer height was 0.25 mm, the line spacing was 2 mm, the size was 10 mm × 10 mm × 2.5 mm, the printing nozzle temperature was 15-25 ° C, the printing platform temperature was 12-17 ° C, the printing speed was 1-3 mm / s, and the extrusion speed was 0.3-0.4 mm. 3 / s, the printed model is irradiated with 405nm blue light at 80-120mW / cm 2 The 3D bioprinted bladder patch was obtained by irradiation and cross-linking for 20 to 40 seconds.

[0073] Preferably, the 3D printing bio-ink is added to the syringe barrel, the air is exhausted, and the 3D bio-printer is connected. The printing parameters are: the inner diameter of the printing needle is 0.25 mm, the printing layer height is 0.25 mm, the line spacing is 2 mm, the size is 10 mm × 10 mm × 2.5 mm. The printing nozzle temperature is 20 ° C, the printing platform temperature is 15 ° C, the printing speed is 2 mm / s, and the extrusion speed is 0.35 mm 3 / s, the printed model was irradiated with 405nm blue light at 100mW / cm 2 The 3D bioprinted bladder patch was obtained by irradiation and cross-linking for 30 seconds.

[0074] The fourth aspect of the present invention is to provide a 3D bioprinted bladder patch prepared according to the preparation method of the 3D bioprinted bladder patch described in the third aspect of the present invention.

[0075] The fifth aspect of the present invention is to provide an application of the 3D bioprinted bladder patch described in the fourth aspect of the present invention, and the 3D bioprinted bladder patch can be used as a biological scaffold.

[0076] Example

[0077] The present invention is further described below by means of specific examples. These embodiments are only intended to illustrate the present invention but are not intended to limit the scope of the present invention.

[0078] Example 1

[0079] 1) Preparation of bladder acellular matrix: bladder tissue frozen at -80°C was thawed at room temperature (e.g. Figure 1 As shown in the figure, first remove part of the tissue at the top of the bladder to change the flow direction of the perfusion solution. The bladder tissue removed from the top of the bladder is connected to the perfusion decellularization system, with the bilateral ureters as the inflow channel of the perfusion solution and the notch at the top of the bladder as the outflow channel of the perfusion solution. First, use 1% Triton X-100 as the perfusion solution for 6 hours, then use deionized water for 1 hour, and then use 1% SDS for 2 hours. Finally, rinse with deionized water for 24 hours to completely remove the chemical detergent to obtain the bladder decellularized matrix, as shown in the figure. Figure 1 shown.

[0080] 2) Preparation of photo-crosslinked bladder decellularized matrix hydrogel: The prepared bladder decellularized matrix was freeze-dried, and 100 ml of hydrochloric acid pepsin solution (1 mg / ml) with a pH of 1 was prepared. Then, 1 g of bladder decellularized matrix freeze-dried powder was added, and the digestion was continued at room temperature of 25°C for 48 hours to prepare a bladder decellularized matrix digestion solution, and the pH was adjusted to 8-9 using a sodium hydroxide solution (1 mol / L). 0.6 g of methacrylic anhydride was added to the bladder decellularized matrix digestion solution at 4°C, and the reaction was continued under stirring for 24 hours under light-proof conditions. During the reaction, the pH was maintained at 8-9, and a 12-14 KDa dialysis membrane was used for continuous dialyzation at 4°C for 2 days, followed by overnight at -80°C and freeze drying in a freeze dryer for 2-3 days to obtain methacrylylated bladder decellularized matrix powder.

[0081] 3) 0.1 g of methacrylated bladder decellularized matrix powder was dissolved in 10 ml of PBS (phosphate buffered saline) solution containing 0.25% (mass concentration) of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) to obtain a photo-crosslinked bladder decellularized matrix hydrogel precursor solution, such as Figure 3 As shown, a photo-crosslinked bladder decellularized matrix hydrogel can be obtained under 405 nm wavelength light for 30 seconds. The state of the hydrogel before and after light exposure is shown in FIG. Figure 4 shown.

[0082] Example 2

[0083] 1) Preparation of bladder decellularized matrix: Thaw the bladder tissue frozen at -80℃ at room temperature, and first remove part of the tissue at the top of the bladder to change the flow direction of the perfusion solution. The bladder tissue removed from the top of the bladder is connected to the perfusion decellularization system, with the bilateral ureters as the inflow channel of the perfusion solution and the notch at the top of the bladder as the outflow channel of the perfusion solution. First, use 0.5% Triton X-100 as the perfusion solution for 7 hours, then use deionized water for 0.5 hours, and then use 0.5% SDS for 2 hours. Finally, rinse with deionized water for 20 hours to completely remove the chemical detergent to obtain the bladder decellularized matrix.

[0084] 2) Preparation of photo-crosslinked bladder decellularized matrix hydrogel: The prepared bladder decellularized matrix was freeze-dried, and 120 ml of hydrochloric acid pepsin solution (0.5 mg / ml) with a pH of 1 was prepared. Then, 1 g of bladder decellularized matrix freeze-dried powder was added, and the digestion was continued at room temperature of 25°C for 45 h to prepare bladder decellularized matrix digestion solution, and the pH was adjusted to 8-9 using sodium hydroxide solution (1 mol / L). 0.5 g of methacrylic anhydride was added to the bladder decellularized matrix digestion solution at 0°C, and the reaction was continued under stirring for 20 h under light-proof conditions. During the reaction, the pH was maintained at 8-9, and a 12-14 KDa dialysis membrane was used for continuous dialyzing at 0°C for 1 day, followed by overnight at -80°C and freeze drying in a freeze dryer for 2-3 days to obtain methacrylylated bladder decellularized matrix powder.

[0085] 3) 0.1 g of methacrylylated bladder decellularized matrix powder was dissolved in 15 ml of PBS (phosphate buffered saline) solution containing 0.2% (mass concentration) of phenyl-2,4,6-trimethylbenzoyl phosphinate lithium (LAP) to obtain a photo-crosslinked bladder decellularized matrix hydrogel precursor solution, and the photo-crosslinked bladder decellularized matrix hydrogel was obtained under light of 405 nm wavelength for 20 s.

[0086] Example 3

[0087] 1) Preparation of bladder decellularized matrix: Thaw the bladder tissue frozen at -80℃ at room temperature, and first remove part of the tissue at the top of the bladder to change the flow direction of the perfusion solution. The bladder tissue removed from the top of the bladder is connected to the perfusion decellularization system, with the bilateral ureters as the inflow channel of the perfusion solution and the notch at the top of the bladder as the outflow channel of the perfusion solution. First, use 2% Triton X-100 as the perfusion solution for 4 hours, then use deionized water for 2 hours, and then use 2% SDS for 0.5 hours. Finally, rinse with deionized water for 30 hours to completely remove the chemical detergent to obtain the bladder decellularized matrix.

[0088] 2) Preparation of photo-crosslinked bladder decellularized matrix hydrogel: The prepared bladder decellularized matrix was freeze-dried, and 80 ml of hydrochloric acid pepsin solution (2 mg / ml) with a pH of 1 was prepared. Then, 1 g of bladder decellularized matrix freeze-dried powder was added, and the digestion was continued at room temperature of 25°C for 50 h to prepare a bladder decellularized matrix digestion solution, and the pH was adjusted to 8-9 using a sodium hydroxide solution (1 mol / L). 0.7 g of methacrylic anhydride was added to the bladder decellularized matrix digestion solution at 5°C, and the reaction was continued with stirring for 30 h under light-proof conditions. During the reaction, the pH was maintained at 8-9, and a 12-14 KDa dialysis membrane was used for continuous dialyzation at 5°C for 2 days, followed by overnight at -80°C and freeze drying in a freeze dryer for 2-3 days to obtain methacrylylated bladder decellularized matrix powder.

[0089] 3) 0.1 g of methacrylylated bladder decellularized matrix powder was dissolved in 5 ml of PBS (phosphate buffered saline) solution containing 0.3% (mass concentration) of phenyl-2,4,6-trimethylbenzoyl phosphinate lithium (LAP) to obtain a photo-crosslinked bladder decellularized matrix hydrogel precursor solution, and the photo-crosslinked bladder decellularized matrix hydrogel was obtained under light of 405 nm wavelength for 45 s.

[0090] Example 4

[0091] 3D bioprinted bladder patch: 0.5 g of gelatin was added to 10 ml of the photo-crosslinked bladder acellular matrix hydrogel prepared in Example 1 and fully dissolved at 40° C. to obtain 3D printing bio-ink.

[0092] Transfer the bio-ink into the syringe barrel, expel the air, and connect it to the 3D bio-printer (such as Figure 7 As shown in Figure A), printing parameters: the inner diameter of the printing needle is 0.25mm, the printing layer height is 0.25mm, the line spacing is 2mm, the size is 10mm×10mm×2.5mm. The printing nozzle temperature is 20℃, the printing platform temperature is 15℃, the printing speed is 2mm / s, and the extrusion speed is 0.35mm 3 / s, photos of bio-ink extruded into filaments in a 3D bio-printer Figure 7 As shown in Figure B, the printed model is irradiated with 405nm blue light at 100mW / cm 2 The 3D bioprinted bladder patch was obtained by irradiation and cross-linking for 30 seconds. The photo of the printed bladder patch is shown in Figure 7 As shown in Figures C and D, the bladder decellularized matrix hydrogel of the present invention can be used for 3D printing of circular or rectangular bladder patches.

[0093] Experimental example

[0094] Experimental Example 1 Thickness Detection and Quantitative Analysis of Bioactive Components of Bladder Decellularized Matrix

[0095] The bladder wall thickness of the bladder tissue before and after decellularization in Example 1 was tested, and the test results are as follows: Figure 2 As shown in Figure B; the DNA content, collagen content, and GAG content of the bladder tissue before and after decellularization in Example 1 were tested, and the test results were as follows: Figure 2 As shown in Figures C, D and E.

[0096] from Figure 2 As can be seen from Figure B, the bladder wall thickness of the decellularized bladder tissue is reduced, but the thickness reduction value is small.

[0097] from Figure 2As shown in Figures C, D, and E, the DNA content of the bladder tissue after decellularization is greatly reduced, the collagen content of the bladder tissue after decellularization is basically unchanged, only slightly reduced, and the GAG ​​content of the bladder tissue after decellularization is basically unchanged. This shows that the bladder decellularized matrix prepared by the decellularization method of the present invention not only removes the cellular components that cause immune response, but also well retains the collagen structure and bioactive components.

[0098] Experimental Example 2 Rheological Characterization Test

[0099] The rheological properties of the bladder decellularized matrix hydrogel, gelatin and bio-ink prepared in Example 1 were tested respectively. The viscosity curves were as follows: Figure 5 The measured temperature scanning curve is shown in Figure 6 shown.

[0100] from Figure 5 It can be seen that under the initial conditions, the viscosity of the bladder decellularized matrix hydrogel prepared in Example 1 is the largest, and the viscosity of gelatin is the smallest. As the shear rate increases, the viscosities of the bladder decellularized matrix hydrogel, gelatin, and bio-ink gradually decrease, and the viscosity of the bio-ink decreases the least. This indicates that the bladder decellularized matrix hydrogel prepared in Example 1 and the bio-ink prepared in Example 4 both have the characteristics of shear thinning.

[0101] from Figure 6 It can be seen that when the temperature is 4°C, the viscosity of the bladder decellularized matrix hydrogel, gelatin, and bio-ink is the largest, and they are in a solid gel state. As the temperature increases, the viscosity of the bladder decellularized matrix hydrogel and bio-ink gradually decreases, indicating that both the bladder decellularized matrix hydrogel and bio-ink have good temperature responsiveness, indicating that the bio-ink is a good 3D bioprinting material, and a bladder patch can be printed using a 3D bioprinter.

[0102] The present invention has been described in detail above in conjunction with specific implementations and exemplary examples, but these descriptions cannot be understood as limiting the present invention. Those skilled in the art understand that, without departing from the spirit and scope of the present invention, a variety of equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its implementation methods, all of which fall within the scope of the present invention. The scope of protection of the present invention shall be subject to the attached claims.

Claims

1. A bladder decellularized matrix hydrogel, characterized in that: The bladder decellularized matrix hydrogel is prepared by methacrylylation of the bladder decellularized matrix and a photoinitiator; The mass ratio of the methacrylylated bladder decellularized matrix to the photoinitiator is 1:(0.2-0.3).

2. The bladder decellularized matrix hydrogel according to claim 1, characterized in that: The methacrylated bladder decellularized matrix is ​​prepared from the bladder decellularized matrix and methacrylic anhydride; The mass ratio of the bladder decellularized matrix to methacrylic anhydride is 1:(0.5-0.7).

3. The bladder decellularized matrix hydrogel according to claim 1, characterized in that: The photoinitiator is selected from one or more of phenyl-2,4,6-trimethylbenzoylphosphinate lithium, Irgacure 2959, and 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone.

4. A method for preparing the bladder decellularized matrix hydrogel according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: The photoinitiator is dissolved in a solvent to obtain a photoinitiator solution, the methacrylylated bladder decellularized matrix is ​​added to the photoinitiator solution to obtain a photo-crosslinked bladder decellularized matrix hydrogel precursor solution, and then the solution is placed under light conditions for crosslinking reaction to obtain a bladder decellularized matrix hydrogel.

5. The preparation method according to claim 4, characterized in that: The method for preparing the methacrylylated bladder decellularized matrix comprises the following steps: The bladder decellularized matrix is ​​freeze-dried to obtain freeze-dried powder of the bladder decellularized matrix. The freeze-dried powder of the bladder decellularized matrix is ​​added to a hydrochloric acid pepsin solution for digestion, and then an alkaline solution is added to adjust the pH. After cooling, methacrylic anhydride is added, and the reaction is stirred under light-proof conditions. Finally, the dialysis is performed with a dialysis membrane and freeze-dried to obtain a methacrylylated bladder decellularized matrix.

6. The preparation method according to claim 5, characterized in that: The method for preparing the bladder decellularized matrix comprises the following steps: The bladder tissue frozen at -80°C was thawed at room temperature, part of the tissue at the top of the bladder was removed and connected to the perfusion decellularization system, the bilateral ureters were used as the inflow channel of the perfusion solution, and the notch at the top of the bladder was used as the outflow channel of the perfusion solution. Triton X-100 with a mass fraction of 0.5-2% was used as the perfusion solution for 4-7 hours, then deionized water was used for perfusion for 0.5-2 hours, and then SDS with a mass fraction of 0.5-2% was used for perfusion for 0.5-2 hours. Finally, it was rinsed with deionized water for 20-30 hours to completely remove the chemical detergent to obtain the bladder decellularized matrix.

7. A method for preparing a 3D bioprinted bladder patch, characterized in that: The preparation method comprises the following steps: Step 1, adding gelatin to the bladder acellular matrix hydrogel according to any one of claims 1 to 3, mixing and dissolving, to obtain 3D printing bio-ink; Step 2: Add the 3D printing bio-ink into the 3D bio-printer and perform 3D printing to obtain a 3D bio-printed bladder patch.

8. The preparation method according to claim 7, characterized in that: In step 1, Gelatin is added to the bladder decellularized matrix hydrogel, the mass ratio of the bladder decellularized matrix hydrogel to gelatin is 10:(0.4-0.7), and it is dissolved at 35-45° C. to obtain 3D printing bio-ink.

9. A 3D bioprinted bladder patch prepared according to the method for preparing a 3D bioprinted bladder patch according to claim 7 or 8.

10. An application of the 3D bioprinted bladder patch according to claim 9, characterized in that: The 3D bioprinted bladder patch can be used as a biological scaffold.

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