A pro-vascularizing dual-network dynamic bio-ink

Abstract

The application discloses a kind of double-network dynamic bio-ink of promoting vascularization. It is composed of PBS buffer, non-dynamic hydrogel component and its crosslinking agent, dynamic hydrogel component and its crosslinking agent, living cells;Among them, the non-dynamic hydrogel component is methyl methacrylate anhydride chemically modified gelatin;Its crosslinking agent is lithium phenyl-2,4,6-trimethyl benzoyl phosphinic acid;The dynamic hydrogel component is the gelatin GelADH modified by adipic acid dihydrazide;Its crosslinking agent is the dextran molecule oxidized by sodium metaperiodate and thus forms aldehyde group.The application optimizes the ratio of non-dynamic hydrogel component and dynamic hydrogel component, and the addition amount of its corresponding crosslinking agent, constructs the double-network dynamic bio-ink with excellent printing performance, and has stability and dynamics, which is a new hydrogel material design scheme that supports dynamic vascular morphogenesis and has printing property simultaneously.

Description

Technical Field

[0001] This invention belongs to the field of vascularized tissue engineering technology, specifically relating to a dual-network dynamic bio-ink that promotes vascularization. Background Technology

[0002] Tissue engineering is a biotechnology aimed at repairing, reconstructing, and regenerating tissues / organs with specific physiological functions. One of its key challenges is achieving vascularization of these tissues. Hydrogels are an important and commonly used material in tissue engineering, providing physical and biochemical factors to simulate the tissue microenvironment required for vascular morphogenesis. Common hydrogel biomaterials are prepared from natural, synthetic, and composite materials through chemical modification. They typically achieve gel formation from aqueous solutions by forming irreversible and stable crosslinks between the functional groups modified in the material. Modified gelatin hydrogels (such as gelatin GelMA modified with methacrylic anhydride) possess cell adhesion sites and can be degraded by cells, often serving as 3D culture scaffolds for vascular endothelial cells. Their formability can also be used as an engineering property in biomanufacturing technologies such as bioprinting to construct biomimetic vascularized tissues with specific shapes and sizes.

[0003] Currently, two types of hydrogels have been used for 3D culture of vascular endothelial cells:

[0004] The first type consists of conventional hydrogels that have undergone stable covalent cross-linking, exhibiting excellent printability, shape fidelity, and structural stability that supports long-term 3D culture. However, cells must actively degrade these conventional hydrogels to achieve their spreadable cell morphology, migrate, and remodel the hydrogel microenvironment, which delays and hinders a range of important cellular behaviors.

[0005] The second type of reversibly cross-linked dynamic hydrogels can promote endothelial cell spreading, migration, and network formation by enhancing matrix-cell interactions. Nevertheless, dynamic hydrogels are prone to degradation due to the presence of reverse reactions in the cross-linking process, thus lacking the stability to support long-term 3D culture.

[0006] Optimizing the design of hydrogel materials to support dynamic vascular morphogenesis while also possessing good printability and other engineering properties is a key issue for their application in vascularized tissue engineering. Summary of the Invention

[0007] The purpose of this invention is to provide a dual-network hydrogel system with complementary stability and dynamism, representing a novel design approach for hydrogel materials that supports dynamic vascular morphogenesis while also offering printability. This invention employs a dual-network hydrogel design strategy, creating a hydrogel system that contains both a dynamic hydrogel network supporting a high degree of vascular morphogenesis and a conventional, stably cross-linkable, non-dynamic hydrogel network. Both hydrogel networks in this system are based on the chemical modification of gelatin molecules.

[0008] The dual-network hydrogel system provided by the present invention comprises non-dynamic hydrogel components and dynamic hydrogel components as raw materials, wherein the mass ratio of non-dynamic hydrogel components to dynamic hydrogel components can be 2-5:2-5, preferably 4:2-5, and more preferably 4:2-3.

[0009] The non-dynamic hydrogel component is methacrylic anhydride-modified gelatin (GelMA) with a modification rate of 20%-80%.

[0010] The dynamic hydrogel component is gelatin GelADH modified with adipic acid dihydrazide (ADH), with a modification rate of 20%-80%.

[0011] The raw materials for preparing the dual-network hydrogel system also include a crosslinking agent for crosslinking the methacrylic anhydride-modified gelatin (GelMA) and a crosslinking agent for crosslinking the adipic dihydrazide (ADH)-modified gelatin (GelADH).

[0012] The crosslinking agent used to crosslink the chemically modified methacrylic anhydride gelatin (GelMA) can be phenyl-2,4,6-trimethylbenzoyl lithium phosphine oxide (LAP), which generates free radicals by initiating the crosslinking of the chemically modified methacrylic anhydride gelatin (GelMA) with blue light.

[0013] The crosslinking agent used to crosslink the gelatin GelADH modified with adipic dihydrazide (ADH) can specifically be a dextran molecule DexCHO that has been oxidized with sodium periodate to form an aldehyde group, with a modification rate of 20%-80%.

[0014] In the dual-network hydrogel system, the mass ratio of gelatin modified with adipic dihydrazide (ADH) to the crosslinking agent used for crosslinking can be 10-20:1.

[0015] The mass ratio of chemically modified methacrylic anhydride gelatin (GelMA) to the crosslinking agent used for its crosslinking can be 2-5:0.125, specifically 4:0.125.

[0016] This invention also provides a modified gelatin dual-network dynamic bio-ink.

[0017] The modified gelatin dual-network dynamic bio-ink provided by this invention is composed of PBS buffer, non-dynamic hydrogel components and their cross-linking agents, dynamic hydrogel components and their cross-linking agents, and living cells.

[0018] The non-dynamic hydrogel component is methacrylic anhydride-modified gelatin (GelMA) with a modification rate of 20%-80%; its crosslinking agent is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP).

[0019] The dynamic hydrogel component is gelatin GelADH modified with adipic dihydrazide (ADH), with a modification rate of 20%-80%; its crosslinking agent is dextran molecules that have been oxidized with sodium periodate (NaIO4) to form aldehyde groups, with a modification rate of 20%-80%.

[0020] In the modified gelatin dual-network dynamic bio-ink, the concentration of the non-dynamic hydrogel component is 1%-10% wt / v, preferably 2%-5% wt / v, and more preferably 4% wt / v;

[0021] The concentration of the dynamic hydrogel component is 1%-10% wt / v, preferably 2%-5% wt / v, and more preferably 2-3% wt / v;

[0022] The mass ratio of the dynamic hydrogel component (gelatin modified with adipic dihydrazide (ADH)) to the crosslinking agent used for its crosslinking can be 10-20:1;

[0023] The concentration of the crosslinking agent used for crosslinking non-dynamic hydrogel components is 0.1-1% wt / v, specifically 0.125% wt / v.

[0024] The present invention also provides a method for constructing vascularized tissue.

[0025] The method for constructing vascularized tissue by bio-3D printing provided by the present invention includes the following steps: mixing the non-dynamic hydrogel component and the dynamic hydrogel component with a corresponding crosslinking agent and pre-embedding live cells therein; casting the resulting hydrogel material system into a micromold or well plate; crosslinking; and 3D culture; or loading the hydrogel system as bio-ink into an extrusion-type bio-3D printer, printing, and then 3D culture to obtain vascularized tissue.

[0026] Vascularized tissue constructed by the above method also falls within the scope of protection of this invention.

[0027] This invention constructs a dual-network dynamic bio-ink with excellent printing performance by optimizing the ratio of non-dynamic hydrogel components and dynamic hydrogel components and the corresponding amount of crosslinking agent added. It combines stability and dynamism, and is a new hydrogel material design scheme that supports dynamic blood vessel morphogenesis while also being printable. Attached Figure Description

[0028] Figure 1 This invention provides a 3D printing process for using a dual-network gelatin dynamic hydrogel system containing vascular endothelial cells as a bio-ink.

[0029] Figure 2 This indicates that without GelMA (a), the formability is poor, while with GelMA (b), the formability is better.

[0030] Figure 3 To optimize printing performance by adjusting the concentration of the dual-network dynamic hydrogel components.

[0031] Figure 4 To compare the stress relaxation characteristics of GelMA and dual-network dynamic hydrogels with the same hardness, the dual-network dynamic hydrogel was prepared by adding an equal volume of 4% wt / v GelMA and its initiator (LAP 0.125% wt / v) to 3% wt / v GelADH hydrogel (DexCHO 0.15% wt / v) (solvent PBS) and then curing it. Its stress relaxation characteristics have a faster time scale than those of GelMA with a fixed concentration of 4.7% wt / v.

[0032] Figure 5 To compare the degree of activation of biochemical signaling pathways required for vascular endothelial cell morphogenesis by GelMA and dual-network dynamic hydrogels of the same stiffness, the dual-network dynamic hydrogel was prepared by adding an equal volume of 4% wt / v GelMA and its initiator (LAP 0.125% wt / v) to 3% wt / v GelADH hydrogel (DexCHO).

[0033] Prepared by curing in 0.15% wt / v (solvent PBS), it activates the mechanotransduction signaling pathway required for endothelial cell morphogenesis to a greater extent than 4.7% wt / v GelMA.

[0034] Figure 6 To compare the effects of pure GelMA hydrogel and dual-network dynamic hydrogel on vascular endothelial cell morphogenesis under co-culture conditions with human fibroblasts.

[0035] Figure 7 To cultivate a dual-network dynamic hydrogel system containing human endothelial cells and human lung fibroblasts after bio-3D printing, and to observe the spreading and connected 3D vascular network by immunofluorescence staining. Detailed Implementation

[0036] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0038] Non-dynamic hydrogel components and dynamic hydrogel components are mixed with corresponding crosslinking agents and pre-loaded with living cells (vascular endothelial cells). The resulting hydrogel material system is loaded as bio-ink into an extrusion-type bio-3D printer, printed, photocured, and 3D cultured to obtain vascularized tissue.

[0039] The non-dynamic hydrogel component is methacrylic anhydride-modified gelatin (GelMA) with a modification rate of 20%-80%; its crosslinking agent is lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid (LAP).

[0040] The dynamic hydrogel component is gelatin GelADH modified with adipic dihydrazide (ADH), with a modification rate of 20%-80%; its crosslinking agent is dextran molecules that have been oxidized with sodium periodate (NaIO4) to form aldehyde groups, with a modification rate of 20%-80%.

[0041] Its process is as follows Figure 1 As shown.

[0042] The GelMA used in the following examples, with a modification rate of 53%, was purchased from SUNP Gel G1 by SUNP Bio-Tech Co., Ltd. (Beijing).

[0043] The GelADH in the following examples has a modification rate of 29.6%, and the preparation method is as follows:

[0044] 1 g of gelatin was dissolved at 10°C in 10 mM pH 5.5 PBS at a concentration of 10%, and the solution was stirred. 1.74 g of adipic hydrazide (ADH, Aladdin) was then added to the solution. Next, 0.77 g of 1-hydroxybenzotriazole (HOBt) and 0.77 g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) were dissolved separately in 10 mL of a 1:1 mixture of DMSO and water, and added dropwise to the reaction system. After adjusting the pH to 5.2, the reaction was maintained at 50°C and stirred for 12 hours. After adjusting the pH to 7.4, the solution was diluted with three portions of deionized water, and then dialyzed against deionized water (MWCO 12 kDa) at 40°C for 3 days, changing the water 5 times daily, followed by lyophilization for 3 days.

[0045] The dextran molecule DexCHO in the following examples, which is oxidized with sodium periodate (NaIO4) to form an aldehyde group, has a modification rate of 19.4%. The preparation method is as follows:

[0046] 1 gram of dextran (Mn = 110,000) was dissolved in 1% distilled water, and then 1 gram of sodium periodate was added to the solution. The solution was then stirred for 0.5 hours in the dark, followed by the addition of 1.0 mL of ethylene glycol to terminate the oxidation reaction and stirring for another 0.5 hours. The solution (MWCO 3500) was then dialyzed for 3 days, with the water changed 5 times a day, and then lyophilized for 3 days.

[0047] Example 1: Rheological characterization of the crosslinking process of two components in a dual-network dynamic hydrogel system under individual or blended conditions.

[0048] An equal volume of 4% wt / v GelMA and its initiator (LAP 0.125% wt / v) was added to 3% wt / v GelADH hydrogel (DexCHO 0.15% wt / v) (solvent PBS), and cured. Hydrogel formation tests demonstrated that this blending method is beneficial for improving the stability of the dynamic hydrogel GelADH. Figure 2 As shown. Figure 2 This indicates that without GelMA (a), the formability is poor, while with GelMA (b), the formability is better.

[0049] Example 2: Optimization of printing performance of hydrogel system

[0050] Printability characterization was performed on a dual-network dynamic hydrogel system containing two components. The concentrations of the two components and their crosslinking agent were adjusted, and printing parameters were optimized for printability testing to screen out the optimal concentration of the dual-network dynamic hydrogel system for bio-3D printing. The results are as follows: Figure 3As shown. A PBS system was used, with LAP concentration of 0.125% wt / v, GelMA concentration of 4% wt / v, and GelADH concentrations of 2% wt / v, 3% wt / v, 4% wt / v, and 5% wt / v, respectively. The mass ratios of GelADH to its corresponding crosslinking agent (DexCHO) were 10:1, 15:1, and 20:1, respectively.

[0051] from Figure 3 It can be seen that GelMA concentration of 4% wt / v, GelADH concentrations of 2% wt / v and 3% wt / v, and mass ratios of GelADH to its corresponding crosslinking agent (DexCHO) of 10:1, 15:1, and 20:1, respectively, achieve equivalently excellent printability. The method for judging printability excellence refers to the printability test published by Ouyang et al. (Ouyang, Liliang et al. “Effect of bioink properties on printability and cellviability for 3D bioplotting of embryonic stem cells.” Biofabrication vol.8,3035020.16Sep.2016,doi:10.1088 / 1758-5090 / 8 / 3 / 035020):

[0052]

[0053] Where C is the roundness of the square pores formed by the hydrogel filaments after printing, and Pr is the printability score. The closer Pr is to 1, the better the printability.

[0054] Example 3: Stress relaxation tests were performed on the dual-network dynamic hydrogel system of the blended components and pure GelMA.

[0055] The dual-network dynamic hydrogel consisted of 4% wt / v GelMA and its initiator (LAP 0.125% wt / v), 3% wt / v GelADH hydrogel (containing 0.15% wt / v dextran molecules oxidized by sodium periodate (NaIO4) to form aldehyde groups) (solvent PBS), and pure GelMA hydrogel with a concentration of 4.7% wt / v and its initiator (LAP).

[0056] (0.125% wt / v). The stress relaxation characteristics of the obtained dual-network dynamic hydrogel system were investigated, and the results are as follows: Figure 4 As shown. By Figure 4 It can be seen that, based on a fixed concentration of GelMA and GelADH, DexCHO, the stress relaxation characteristics are faster than those of pure GelMA hydrogel at the same hardness.

[0057] Example 4: Detection of the activation level of the mechanical transduction signaling pathway of endothelial cells by the dual-network dynamic hydrogel system of the blended components and pure GelMA.

[0058] The dual-network dynamic hydrogel consisted of 4% wt / v GelMA and its initiator (LAP 0.125% wt / v), 3% wt / v GelADH hydrogel (containing 0.15% wt / v dextran molecules oxidized by sodium periodate (NaIO4) to form aldehyde groups) (solvent PBS), and pure GelMA hydrogel with a concentration of 4.7% wt / v and its initiator (LAP).

[0059] Endothelial cells were embedded at a density of 4,000,000 cells / mL at this concentration (0.125% wt / v). On day three, key characteristic proteins of mechanotransduction, such as integrinβ1, MMP14, and p-FAK, were quantitatively characterized using fluorescence. The results demonstrated that the dual-network dynamic hydrogel system of the blend components activated mechanotransduction in endothelial cells to a greater extent, which was beneficial to endothelial cell morphogenesis. Figure 5 (As shown).

[0060] Example 5: Detection of the effects of the dual-network dynamic hydrogel system of the blended components and pure GelMA hydrogel on endothelial cell morphogenesis under co-culture conditions with human fibroblasts.

[0061] The dual-network dynamic hydrogel consisted of 4% wt / v GelMA and its initiator (LAP 0.125% wt / v), 3% wt / v GelADH hydrogel (containing 0.15% wt / v dextran molecules oxidized by sodium periodate (NaIO4) to form aldehyde groups) (solvent PBS), and pure GelMA hydrogel at concentrations of 4.7% wt / v and its initiator (LAP 0.125% wt / v), and 4% wt / v and its initiator (LAP 0.125% wt / v). Cells were embedded at these concentrations at densities of 4,000,000 endothelial cells / mL and 2,000,000 human lung fibroblasts / mL. Immunofluorescence morphological characterization of the embedded cells was performed on day 3. The results demonstrated that the blended dual-network dynamic hydrogel system facilitated greater extension and network formation of endothelial cells and fibroblasts, which was beneficial for endothelial cell morphogenesis. Figure 6 (As shown).

[0062] Example 6: Construction of a 3D network structure of vascularized tissue based on a cell-loaded hydrogel system

[0063] An equal volume of 4% wt / v GelMA and its initiator (LAP 0.125% wt / v) was added to 3% wt / v GelADH hydrogel (containing 0.15% wt / v dextran molecules oxidized to aldehyde groups via sodium periodate oxidation) (solvent PBS). This mixture was loaded into the syringe of a 3D bioprinter. The printing temperature was set to 12 degrees Celsius, and a grid-like structure was printed according to the printer's built-in program. After printing, the structure was cured using UV light. Culture medium was added, and the mixture was incubated for 7 days at 37 degrees Celsius with 5% CO2.

[0064] A dual-network dynamic hydrogel system containing human endothelial cells and human lung fibroblasts was cultured for 7 days after bio-3D printing, and the spreading and connected 3D vascular network was observed by immunofluorescence staining. The results are as follows: Figure 7 As shown. By Figure 7 It can be seen that a 3D network structure with vascular endothelial cells spreading and interconnected was obtained by culturing the cell-loaded hydrogel system for 7 days.

[0065] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A modified gelatin dual-network dynamic bio-ink, comprising PBS buffer, a non-dynamic hydrogel component and its cross-linking agent, a dynamic hydrogel component and its cross-linking agent, and live cells; in, The non-dynamic hydrogel component is methacrylic anhydride-modified gelatin (GelMA) with a modification rate of 20%-80%; its crosslinking agent is lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid. The dynamic hydrogel component is gelatin GelADH modified with adipic acid dihydrazide, with a modification rate of 20%-80%; its crosslinking agent is dextran molecules that have been oxidized with sodium periodate to form aldehyde groups, with a modification rate of 20%-80%; in the modified gelatin dual-network dynamic bio-ink, the concentration of the non-dynamic hydrogel component is 4%wt / v. The concentration of the dynamic hydrogel component is 2%-3%wt / v; The mass ratio of the dynamic hydrogel component to the crosslinking agent used for its crosslinking is 10-20:1; The concentration of the crosslinking agent used to crosslink the non-dynamic hydrogel component is 0.125% wt / v.

2. A method for constructing vascularized tissue, comprising the following steps: pre-embedding live cells in a dual-network hydrogel system, casting the resulting hydrogel material system into a micromold or well plate, cross-linking, 3D culture, or loading the hydrogel system as bio-ink into an extrusion-type bio-3D printer, printing and then 3D culture to obtain vascularized tissue; The dual-network hydrogel system includes a non-dynamic hydrogel component and its crosslinking agent, and a dynamic hydrogel component and its crosslinking agent; in, The non-dynamic hydrogel component is methacrylic anhydride-modified gelatin (GelMA) with a modification rate of 20%-80%; its crosslinking agent is lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid. The dynamic hydrogel component is gelatin (GelADH) modified with adipic acid dihydrazide, with a modification rate of 20%-80%; its crosslinking agent is dextran molecules that have been oxidized with sodium periodate to form aldehyde groups, with a modification rate of 20%-80%. In the modified gelatin dual-network dynamic bio-ink, the concentration of the non-dynamic hydrogel component is 4%wt / v; The concentration of the dynamic hydrogel component is 2%-3%wt / v; The mass ratio of the dynamic hydrogel component to the crosslinking agent used for its crosslinking is 10-20:1; The concentration of the crosslinking agent used to crosslink the non-dynamic hydrogel component is 0.125% wt / v.

3. Vascularized tissue constructed by the method of claim 2.

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