A myocardial tissue engineering scaffold for promoting myocardial cell spreading and maturation, and a preparation method and application thereof

Abstract

This invention relates to a myocardial tissue engineering scaffold that promotes cardiomyocyte spreading and maturation, its preparation method, and its applications. The scaffold preparation method includes the following steps: mixing and dissolving GelMA, Fibrin, and PBS buffer to obtain a hydrogel precursor solution; adding cardiomyocytes to the hydrogel precursor solution, and solidifying the hydrogel through photocrosslinking and enzymatic crosslinking to obtain myocardial engineered tissue; and sequentially subjecting the myocardial engineered tissue to spreading culture, PEG-cured culture, and swelling culture to obtain the myocardial tissue engineering scaffold. The combination of GelMA and Fibrin imparts suitable stress relaxation properties to the scaffold, and the sequential treatment of spreading-curing-swelling achieves gradual optimization of the scaffold's mechanical properties, making the final mechanical properties of the scaffold match those of natural myocardial tissue. This significantly promotes cardiomyocyte spreading and functional maturation and can be used to prepare myocardial patches, myocardial drug screening models, or cardiac disease models.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering and tissue engineering, specifically relating to a myocardial tissue engineering scaffold that promotes cardiomyocyte spreading and maturation, its preparation method, and its application. Background Technology

[0002] Myocardial tissue engineering aims to construct functional myocardial patches or organoids for drug screening, disease model building, and repair therapy of damaged hearts. Myocardial tissue engineering inks act as a supporting medium for cardiomyocytes during the production of cardiac organoids or myocardial cell-loaded patches, possessing good biocompatibility and a certain degree of molding ability and mechanical strength to maintain the macroscopic structure of the printed material. However, traditional inks generally lack stress relaxation properties that match those of natural myocardial tissue. Photopolymerization 3D printing technology, due to its ability to construct high-precision three-dimensional structures, has been widely used in the fabrication of myocardial tissue engineering scaffolds.

[0003] Chinese patent application CN119971151A discloses a myocardial tissue construct with both biomimetic structure and function and its preparation method. The components of the myocardial tissue construct include: a hydrogel matrix (e.g., methacrylated gelatin GelMA) with rheological properties suitable for 3D printing, a crosslinking agent dispersed in the hydrogel matrix, inorganic biomaterial particles with myocardial protection and angiogenesis promotion bioactivity, cardiomyocytes and angiogenesis-related cells; through bio-3D printing and photo-crosslinking curing, the cardiomyocytes and angiogenesis-related cells are arranged in a regular pattern in three-dimensional space, mimicking the cell distribution pattern in natural myocardial tissue.

[0004] However, in this existing technical solution, the mechanical properties of photocrosslinked hydrogels such as GelMA are mainly derived from the covalent network formed by photocrosslinking. It lacks stress relaxation characteristics that match natural myocardial tissue, making it difficult to meet the needs of myocardial cells for a dynamic mechanical microenvironment, resulting in limited cell spread and insufficient functional maturation.

[0005] Therefore, there is considerable room for improvement in the existing technology. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a myocardial tissue engineering scaffold that promotes cardiomyocyte spreading and maturation, as well as its preparation method and application. This invention imparts suitable stress relaxation properties to the scaffold through a composite of GelMA and Fibrin, and achieves gradual optimization of the scaffold's mechanical properties through a sequential process of spreading-curing-swelling, resulting in scaffold mechanical properties that ultimately match those of natural myocardial tissue.

[0007] To achieve the above objectives, the first aspect of the present invention provides a method for preparing a myocardial tissue engineering scaffold that promotes cardiomyocyte spreading and maturation, comprising the following steps: (1) Mix and dissolve gelatin methacryloyl (GelMA), fibrin and PBS buffer to obtain hydrogel precursor solution; (2) Add cardiomyocytes to the hydrogel precursor solution of step (1), and solidify the hydrogel by photocrosslinking and enzyme crosslinking to obtain myocardial engineered tissue; (3) The myocardial engineered tissue obtained in step (2) is spread and cultured in cell culture medium to spread the myocardial cells; (4) The myocardial engineered tissue after step (3) was spread and cultured in a cell culture medium containing polyethylene glycol (PEG) for solidification culture; (5) The myocardial engineered tissue after solidification culture in step (4) is placed in a cell culture medium without PEG for swelling culture to obtain the myocardial engineered scaffold.

[0008] This invention utilizes a composite of GelMA and Fibrin as the hydrogel matrix. The introduction of Fibrin introduces a dynamic fibrin network into the material based on the photocrosslinked covalent network. Under stress, this network can achieve stress relaxation through chain segment rearrangement and stress dissipation, laying the foundation for subsequent cell spreading and functional maturation. The mechanical properties of the scaffold are progressively optimized through a sequential process of spreading, curing, and swelling: spreading culture provides a sufficient spreading window for cardiomyocytes in a low-modulus state, promoting intercellular junction formation and the establishment of synchronized beating; curing culture increases the crosslinking density through the dehydration and shrinkage effect of PEG, significantly enhancing the scaffold modulus; swelling culture, by placing the scaffold in a conventional culture medium, allows it to moderately absorb water and swell, stabilizing its mechanical strength and ensuring that the scaffold's storage modulus and stress relaxation characteristics are highly matched to those of natural myocardial tissue.

[0009] Preferably, in the hydrogel precursor solution obtained in step (1), the concentration of GelMA is 3-5% (w / v) and the concentration of Fibrin is 2.5-7.5% (w / v).

[0010] The above settings regulate the crosslinking density and network structure of the hydrogel to balance the mechanical support performance and stress relaxation ability of the scaffold. The concentration of GelMA primarily determines the density of the photocrosslinked covalent network: below 3%, the network is too sparse, failing to provide sufficient spatial support and mechanical stability for cells, leading to structural collapse; above 5%, the crosslinking density is too high, restricting polymer chain movement and significantly reducing stress relaxation ability. Fibrin forms a fiber network through enzymatic crosslinking, endowing the material with stress dissipation capabilities and providing cell adhesion sites; below 2.5%, the stress relaxation effect and cell adhesion are insufficient; above 7.5%, the fiber network is too dense, limiting cell spreading space.

[0011] Preferably, the process conditions for photocrosslinking in step (2) are as follows: add a photoinitiator, use 3D printing technology to print layer by layer, and perform photocuring treatment to obtain a one-time cured molded printed object.

[0012] By using 3D printing technology to print layer by layer, a three-dimensional scaffold with a pre-set macroscopic structure and internal pores can be constructed. The initial structural strength of the printed material is given by photopolymerization, providing a structurally complete prototype of engineered myocardial tissue for subsequent culture.

[0013] Preferably, the process conditions for enzyme crosslinking in step (2) are as follows: the obtained molded printed material is placed in thrombin solution for secondary crosslinking to obtain secondary crosslinked myocardial engineered tissue.

[0014] This invention employs a two-stage cross-linking process: photocross-linking imparts initial structural strength to the GelMA network to maintain printing fidelity, while thrombin-based secondary cross-linking forms a fibrin network. This two-stage cross-linking process avoids over-densification or insufficient mechanical strength that can occur with a single cross-linking method, enabling the scaffold to possess both structural stability and dynamic viscoelasticity. This preserves the necessary stress relaxation capacity and fibrin adhesion sites for subsequent cardiomyocyte deployment.

[0015] Preferably, the photoinitiator is LAP, and the photocuring treatment is performed at a wavelength of 450 nm. The final concentration of the photoinitiator is 0.05% (w / v); the concentration of the thrombin is 25 U / mL; and the secondary crosslinking condition is treatment at 37°C for 30 minutes.

[0016] Preferably, the spreading and culturing time in step (3) is 3-5 days. If the culturing time is less than 3 days, the gel network may not have fully recovered its hydration state, and the mechanical properties will still be fluctuating; if the culturing time is longer than 5 days, the mechanical properties have reached a plateau, and further extending the culturing time will have limited effect on improving the strength stability, but will instead prolong the preparation cycle and culturing cost.

[0017] Preferably, the PEG in step (4) has a molecular weight of 2000, a concentration of 6-9% (w / v) in the culture medium, and a curing culture time of 48 hours. When the PEG concentration is below 6%, the curing enhancement effect is not significant, and the modulus increase is insufficient; when it is above 9%, the dehydration effect is too strong, which will lead to excessive network shrinkage, causing the scaffold's stable modulus to exceed the range of natural myocardial tissue, which is not conducive to maintaining a mechanical microenvironment that matches the natural myocardium.

[0018] Preferably, the swelling culture time in step (5) is 3-5 days.

[0019] After PEG curing and culturing, the scaffold is placed in a PEG-free conventional culture medium to achieve sufficient water absorption and swelling. This allows the hydrogel network to gradually recover to a suitable hydration state after the PEG dehydration and shrinkage effect is relieved, enabling the mechanical strength of the scaffold to stabilize in a dynamic equilibrium. When the culturing time is less than 3 days, the PEG is not completely replaced, the internal stress of the network is not fully released, and the mechanical properties remain fluctuating. When the culturing time is longer than 5 days, the mechanical properties have reached a plateau, and further extension has limited effect on improving strength stability. Instead, it prolongs the preparation cycle, increases the risk of contamination, and increases culturing costs.

[0020] A second aspect of the present invention provides a myocardial tissue engineering scaffold obtained by the above preparation method.

[0021] A third aspect of the present invention provides the application of the above-mentioned myocardial tissue engineering scaffold in the preparation of myocardial patches, myocardial drug screening models, or cardiac disease models.

[0022] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention uses a composite of GelMA and Fibrin as a hydrogel matrix. The introduction of Fibrin introduces a dynamic fibrin network into the material on the basis of the photocrosslinked covalent network. Under stress, it can achieve stress relaxation through chain segment rearrangement and stress dissipation, laying the foundation for subsequent cell spreading and functional maturation.

[0023] 2. This invention achieves gradual optimization of the mechanical properties of the scaffold through a sequential process of spreading, solidification, and swelling: spreading culture provides sufficient spreading window for cardiomyocytes in a low-modulus state, promoting the formation of intercellular connections and the establishment of synchronous beating; solidification culture increases the cross-linking density through the dehydration and shrinkage effect of PEG, significantly enhancing the modulus of the scaffold; swelling culture allows the scaffold to absorb water and swell moderately by being placed in a conventional culture medium, and the mechanical strength tends to be balanced and stable, so that the energy storage modulus and stress relaxation characteristics of the scaffold are highly matched with those of natural myocardial tissue.

[0024] 3. The scaffold of this invention significantly promotes the spread and functional maturation of cardiomyocytes and can be used to prepare myocardial patches, myocardial drug screening models, or heart disease models. Attached Figure Description

[0025] Figure 1 The graph shows the stress relaxation ability test results of different formulations in Example 1; Figure 2 This is a diagram showing the spreading effect of cardiomyocytes in different Fibrin concentration groups when the GelMA concentration is 3% in Example 1. Figure 3 This is a staining result of the mitochondrial maturation of cardiomyocytes in different Fibrin concentration groups when the GelMA concentration was 3% in Example 1; Figure 4 The graph shows the changes in the modulus of the natural myocardium and the modulus of the patch at different treatment stages in Example 2; where A represents the range of the storage modulus of the natural myocardial tissue, B represents the modulus change during the 6% PEG curing process, and C represents the modulus change after swelling. Figure 5 The graphs show the changes in stress relaxation capacity of the patch after curing and swelling in Example 2; where A represents the changes in stress relaxation capacity after curing with different PEG concentrations, and B represents the changes in stress relaxation capacity after curing with different PEG concentrations and swelling. Figure 6 The images show the performance evaluation of myocardial patches in different treatment groups in Example 2; where A is immunofluorescence staining (CX43), B is cell beating under white light, and C is the calcium transient detection result. Figure 7 This is a graph showing the efficacy of different treatment groups in a rat model of myocardial infarction after 28 days of treatment, as described in Example 2. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.

[0027] Example 1 A method for preparing a myocardial tissue engineering scaffold that promotes the spreading and maturation of ventricular myocytes in neonatal SD rats includes the following steps: (1) Weigh GelMA and Fibrin powder, then add them to PBS buffer and mix to dissolve, to obtain hydrogel precursor solution; wherein, in the obtained hydrogel precursor solution, the concentration of GelMA is 3% (w / v) and the concentration of Fibrin is 2.5% (w / v). (2) Newborn SD rat ventricular myocytes were subjected to a 1×10 7The cells / mL density was added to the hydrogel precursor solution of step (1), and then the photoinitiator LAP with a final concentration of 0.05% (w / v) was added. The 3D printing technology was used to print layer by layer. After each layer was printed, it was immediately irradiated with 450nm blue light for photocuring treatment to obtain a one-time cured shaped printed material. The obtained shaped printed material was placed in 25U / mL thrombin solution and cross-linked at 37℃ for 30min to obtain myocardial engineered tissue. (3) The myocardial engineered tissue obtained in step (2) was transferred into DMEM medium containing 10% fetal bovine serum and cultured in an incubator at 37°C and 5% CO2 for 3 days. Significant spreading of myocardial cells and spontaneous synchronous beating were observed. (4) The myocardial engineered tissue after step (3) was spread and cultured in DMEM medium containing PEG (containing 10% fetal bovine serum) for 48 hours to solidify and culture; wherein the molecular weight of PEG is 2000 and its concentration in the medium is 6% (w / v). (5) The myocardial engineered tissue after solidification culture in step (4) was placed in DMEM medium (without PEG) containing 10% fetal bovine serum for swelling culture for 3 days to obtain the myocardial engineered scaffold.

[0028] The products prepared in Example 1 at different stages were subjected to phased verification of stress relaxation characteristics, cell spreading effect, and cardiomyocyte maturity.

[0029] 1. Experimental Materials Animals: 15 SD suckling mice aged 1-3 days.

[0030] Main reagents: GelMA (60% cross-linking), bovine fibrin, PEG-2000, thrombin, LAP, DMEM medium, fetal bovine serum, mitochondrial staining reagent.

[0031] 2. Experimental Grouping and Testing Phase 1: After the second crosslinking in step (2) To screen for the optimal stress relaxation formulation, multiple experimental groups were established, including cross-combinations of GelMA (3%, 4%, 5%) and Fibrin (2.5%, 5%, 7.5%): 3% GelMA + 2.5% Fibrin (G3F2.5), 3% GelMA + 5% Fibrin (G3F5), 3% GelMA + 7.5% Fibrin (G3F7.5), 4% GelMA + 2.5% Fibrin (G4F2.5), 4% GelMA + 5% Fibrin (G4F5), 4% GelMA + 7.5% Fibrin (G4F7.5), 5% GelMA + 2.5% Fibrin (G5F2.5), 5% GelMA + 5% Fibrin (G5F5), and 5% GelMA + 7.5% Fibrin (G5F7.5). The stress relaxation ability of each group of hydrogels after secondary cross-linking was tested.

[0032] The cured sample was placed on a rheometer platform, and strains of 5% and 10% were applied respectively. Stress decay was recorded over 400 seconds—the faster and greater the decrease in the curve, the stronger the stress relaxation ability. The stress relaxation ability test results are as follows: Figure 1 As shown, the lower the GelMA concentration, the stronger the stress relaxation ability; the formulation group with 3% GelMA exhibits the strongest stress relaxation ability, indicating that the formulation has suitable dynamic mechanical properties after secondary crosslinking.

[0033] Second stage: After step (3) of spreading and cultivation is completed Based on the stress relaxation results of the first stage, the combination groups of 3% GelMA and Fibrin (2.5%, 5%, 7.5%) were selected, with the 5% GelMA group (i.e., no Fibrin) serving as the control. The dosage was 1×10⁻⁶. 7 Newborn SD rat ventricular myocytes were seeded at a density of cells / mL. After 3 and 5 days of spreading culture, samples were taken for immunofluorescence staining (α-actinin, CX43), and cell spreading morphology and connexin expression were observed under a laser confocal microscope. After 5 days of spreading culture, mitochondrial staining was performed on the samples to observe mitochondrial morphology and distribution, and to identify the degree of cell maturity.

[0034] Cell spreading observation results are as follows Figure 2 As shown, after 3 days of spreading culture, the cells began to spread significantly, exhibiting a spindle-shaped extension; after 5 days of spreading culture, the cells formed extensive cell junctions, CX43 protein expression increased significantly, and myofibrils were arranged in an orderly manner. This indicates that suitable stress relaxation characteristics provided a favorable mechanical microenvironment for cell spreading.

[0035] Mitochondrial staining results as follows Figure 3As shown, the GelMA 3% + Fibrin 2.5% group exhibited the best mitochondrial maturity, indicating that the scaffold of this formulation can effectively maintain the mature phenotype of cardiomyocytes during the spreading culture stage.

[0036] 3. Conclusion The scaffolds prepared using the method in Example 1 exhibit excellent stress relaxation characteristics from the secondary cross-linking stage and demonstrate excellent cell spreading promotion and cardiomyocyte maturation maintenance effects after spreading culture. Among them, the group with 3% GelMA + 2.5% Fibrin showed the best performance.

[0037] Example 2 A method for preparing a myocardial patch includes the following steps: (1) Weigh GelMA and Fibrin powder, then add them to PBS buffer and mix to dissolve, to obtain hydrogel precursor solution; wherein, in the obtained hydrogel precursor solution, the concentration of GelMA is 3% (w / v) and the concentration of Fibrin is 2.5% (w / v). (2) Newborn SD rat ventricular myocytes were subjected to 5×10 6 The cells / mL density was added to the hydrogel precursor solution of step (1), and then the photoinitiator LAP with a final concentration of 0.05% (w / v) was added. The myocardial patch structure of 10mm×10mm×1mm was printed layer by layer using 3D printing technology. After each layer was printed, it was immediately irradiated with 450nm blue light for photocuring treatment to obtain a one-time cured shaped printed material. The obtained shaped printed material was placed in 25U / mL thrombin solution and subjected to secondary cross-linking at 37℃ for 30min. (3) The cross-linked patch from step (2) was transferred into DMEM medium containing 10% fetal bovine serum and cultured in an incubator at 37°C and 5% CO2 for 5 days. Significant spread of cardiomyocytes and spontaneous synchronous beating were observed. (4) The myocardial patch after spreading and culturing in step (3) is solidified and cultured in DMEM medium containing PEG (containing 10% fetal bovine serum) for 48 hours; wherein the molecular weight of PEG is 2000 and its concentration in the medium is 6% (w / v). (5) The myocardial patch after solidification culture in step (4) was placed in DMEM medium (without PEG) containing 10% fetal bovine serum for swelling culture for 3 days to obtain the myocardial patch.

[0038] The products prepared in Example 2 at different stages were subjected to phased verification of stress relaxation characteristics, cell spreading effect, and cardiomyocyte maturity.

[0039] 1. Experimental Materials Animals: 15 1-3 day old SD suckling mice (for extracting ventricular myocardial cells from newborn SD rats); 30 6-week-old male rats were used to establish a myocardial infarction model and evaluate the efficacy in vivo. They were randomly divided into 5 groups (n=6 per group): blank control group, myocardial infarction model group, GelMA patch treatment group, GF patch treatment group, and GF-fixed patch treatment group. Animals were kept at a room temperature of 20-25℃ and a relative humidity of 40-50%, with natural day and night lighting, and free access to food and water.

[0040] Main reagents: GelMA (60% cross-linking), bovine fibrin, PEG-2000, thrombin, LAP, DMEM medium, fetal bovine serum.

[0041] Ventricular tissue was harvested from 1-3 day old SD rats through thoracotomy, minced, digested with trypsin and collagenase, and purified using differential adhesion method to obtain neonatal SD rat ventricular myocytes.

[0042] 2. Continuous testing for dynamic control of mechanical properties To investigate the effect of PEG curing process parameters on the mechanical properties of the patch, samples were taken after completing steps (4) and (5) according to the preparation scheme of Example 2, and continuous dynamic control of mechanical properties was tested. (1.1) Based on the 3% GelMA + 2.5% Fibrin formulation, 6% PEG-2000 was used for curing for 48 hours, 120 hours, and 168 hours respectively (i.e., curing and cultivation in step (4)). Samples were taken and frequency scanning tests were performed using a rheometer to detect the storage modulus (G') and loss modulus (G''). The test results are as follows. Figure 4 As shown in B in the diagram.

[0043] (1.2) Based on the 3% GelMA + 2.5% Fibrin formulation, 6%, 9%, and 12% (w / v) PEG-2000 were used for curing treatment for 48 hours, and after swelling culture for 3 days (i.e., swelling culture in step (5)), samples were taken to test the stable storage modulus. The results are as follows. Figure 4 As shown in C.

[0044] (1.3) Based on the 3% GelMA + 2.5% Fibrin formulation, 0%, 6%, and 9% (w / v) PEG-2000 were used for curing treatment for 48 hours (i.e., step (4) curing culture). Samples were taken and subjected to stress relaxation tests with 5% and 10% strain respectively. The change of relaxation modulus over time was detected. The results are as follows. Figure 5 As shown in A in the diagram.

[0045] (1.4) Based on the 3% GelMA + 2.5% Fibrin formulation, 0%, 6%, and 9% (w / v) PEG-2000 were used for curing treatment for 48 hours (i.e., step (4) curing culture), and after swelling culture for 3 days (i.e., step (5) swelling culture), samples were taken and subjected to stress relaxation tests with 5% and 10% strain respectively. The change of relaxation modulus over time was detected, and the results are as follows. Figure 5 As shown in B in the diagram.

[0046] Figure 4 In this context, A represents the storage modulus range of natural myocardial tissue. Figure 4 As shown in Figure B, the storage modulus of the hydrogel first increases and then stabilizes with the extension of PEG curing time: after 48 hours of curing with 6% PEG, the storage modulus of the hydrogel is significantly improved; after 120 hours of curing with 6% PEG, the storage modulus of the hydrogel further increases; and after 168 hours of curing with 6% PEG, the storage modulus of the hydrogel decreases slightly. Figure 4 As shown in Figure C, the higher the PEG concentration, the higher the stable modulus of the scaffold after swelling: the modulus of the 6% PEG group is moderate, matching the modulus range of natural myocardial tissue; the modulus of the 9% PEG group is relatively high, close to the upper limit of natural myocardial tissue; the modulus of the 12% PEG group is too high, exceeding the range of natural myocardial tissue. Considering both cell compatibility and mechanical compatibility, curing 6% PEG-2000 for 48 hours is the optimal process condition.

[0047] from Figure 5 As can be seen from A, the stress relaxation ability of the patch is enhanced by adding PEG for curing in step (4). Figure 5 As shown in B, the patch can still maintain good stress relaxation properties after swelling culture, which is beneficial to the spread and functional maturation of cardiomyocytes.

[0048] 3. In vitro functional assessment of myocardial patches The myocardial patch (i.e., the G3F2.5+PEG6% group) obtained after completing step (5) according to the preparation method of Example 2 was used as a reference example, with the group without Fibrin in step (1) (i.e., the GelMA group) and the group without PEG in step (4) (i.e., the G3F2.5 group) being used for functional identification: (2.1) Immunofluorescence staining of CX43 / α-actinin was used to assess cell connectivity and maturity. The results are as follows: Figure 6 As shown in A; (2.2) Observe the cell beating under white light, and the results are as follows: Figure 6 As shown in B; (2.3) Calcium transient detection was used to assess cellular electrophysiological function, and the results are as follows: Figure 6 As shown in C.

[0049] from Figure 6 It is evident that the G3F2.5+PEG6% group exhibited the optimal functional phenotype: significantly increased expression of CX43 channel protein and good intercellular electrical coupling (see...). Figure 6 (A) Cardiac cardiomyocytes spread and grow evenly, resulting in synchronous beating (see A). Figure 6 (B in the text); calcium transients have higher frequencies and stronger signals, and their electrophysiological functions are more mature (see...). Figure 6 (C in the middle).

[0050] 4. Validation of the therapeutic effect in an in vivo rat model of myocardial infarction A myocardial infarction model was established in SD rats. Cultured myocardial patches were attached to the infarct area. After 28 days, multi-channel electrophysiological mapping and tissue staining were performed on the rat hearts to evaluate the therapeutic effect.

[0051] Five experimental groups were established: a blank control group (Control), a myocardial infarction model group (Model), treatment group 1: 5% GelMA patch group (GelMA), treatment group 2: 3% GelMA + 2.5% Fibrin patch group (G3F2.5), and treatment group 3: 3% GelMA + 2.5% Fibrin + PEG-cured patch group (G3F2.5 + PEG6%).

[0052] like Figure 7 The efficacy test results of the above five experimental groups are shown. From Figure 7 Multichannel electrophysiological mapping showed that the G3F 2.5 + PEG 6% group significantly improved electrical conduction around myocardial infarction; from Figure 7 Succinimide-eosin staining, Sirius red staining, and Masson staining showed that the infarct area was significantly reduced and the degree of fibrosis was decreased in the G3F2.5+PEG6% group.

[0053] 5. Conclusion Therefore, the myocardial tissue engineering scaffolds (including but not limited to patch morphology) prepared by the method of the present invention are mechanically matched with natural myocardium, have suitable stress relaxation characteristics, and have significant effects on promoting myocardial cell maturation and treating myocardial infarction.

[0054] Furthermore, since the scaffold of the present invention can support cardiomyocytes to maintain a mature functional phenotype (synchronous beating, calcium transient, and high CX43 expression) in vitro, and its mechanical properties can be adjusted to match those of natural myocardium, it can also be used as a three-dimensional culture carrier for myocardial drug screening models or cardiac disease models to evaluate the effect of drugs on the contractile function of cardiomyocytes or to simulate the functional changes of cardiomyocytes under pathological conditions.

[0055] The above description is only a preferred embodiment of the present invention. Therefore, all equivalent changes or modifications made to the structure, features and principles described in the claims of this patent application are included in the scope of this patent application.

Claims

1. A method for preparing a myocardial tissue engineering scaffold that promotes cardiomyocyte spreading and maturation, comprising the following steps: (1) Mix and dissolve GelMA, Fibrin and PBS buffer to obtain hydrogel precursor solution; (2) Add cardiomyocytes to the hydrogel precursor solution of step (1), and solidify the hydrogel by photocrosslinking and enzyme crosslinking to obtain myocardial engineered tissue; (3) The myocardial engineered tissue obtained in step (2) is spread and cultured in cell culture medium to spread the myocardial cells; (4) The myocardial engineered tissue after step (3) was spread and cultured in a cell culture medium containing PEG was then solidified and cultured. (5) The myocardial engineered tissue after solidification culture in step (4) is placed in a cell culture medium without PEG for swelling culture to obtain the myocardial engineered scaffold.

2. The production method according to claim 1, characterized by, In the hydrogel precursor solution obtained in step (1), the concentration of GelMA is 3-5% (w / v) and the concentration of Fibrin is 2.5-7.5% (w / v).

3. The preparation method according to claim 1, characterized in that, The process conditions for photocrosslinking in step (2) are as follows: add a photoinitiator, use 3D printing technology to print layer by layer, and perform photocuring treatment to obtain a one-time cured molded print.

4. The preparation method according to claim 3, characterized in that, The process conditions for enzyme crosslinking in step (2) are as follows: the obtained molded printed material is placed in thrombin solution for secondary crosslinking to obtain secondary crosslinked myocardial engineered tissue.

5. The preparation method according to claim 4, characterized in that, The photoinitiator is LAP, which is photocured at a wavelength of 450 nm. The final concentration of the photoinitiator is 0.05% (w / v). The concentration of the thrombin is 25 U / mL. The secondary crosslinking condition is treatment at 37°C for 30 minutes.

6. The preparation method according to claim 1, characterized in that, The spreading and cultivation time described in step (3) is 3-5 days.

7. The preparation method according to claim 1, characterized in that, The PEG mentioned in step (4) has a molecular weight of 2000, a concentration of 6-9% (w / v) in the culture medium, and a curing time of 48 hours.

8. The preparation method according to claim 1, characterized in that, The swelling culture time described in step (5) is 3-5 days.

9. A myocardial tissue engineering scaffold obtained by the preparation method according to any one of claims 1-8.

10. The use of the myocardial tissue engineering scaffold of claim 9 in the preparation of myocardial patches, myocardial drug screening models, or cardiac disease models.

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