Bioprinted heart valve fibrosis disease model, preparation method therefor, and use thereof

Abstract

Provided are a bioprinted heart valve fibrosis disease model, a preparation method therefor, and use thereof. The method comprises the following steps: using a hydrogel containing valve cells as a printing material, printing by means of digital light processing (DLP) technology to obtain a valve model, wherein the digital light processing technology comprises importing design drawings of different grayscales into a digital light processing printing device, and after printing, culturing to obtain bioprinted heart valve fibrosis disease models with different stiffnesses. According to the method, by means of regulating DLP printing parameters, adjusting the stiffness of an extracellular matrix, controlling the degree of fibrosis of various mechanically responsive cells such as fibroblasts, and simulating different fibrotic states, the effect of the in vivo environment on valve tissue can be better simulated.

Description

A bioprinted model of heart valve fibrosis, its preparation method and application Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a bioprinted model of heart valve fibrosis, its preparation method, and its application. Background Technology

[0002] Heart valve disease is a common cardiovascular disease, and valvular fibrosis is one of the main causes of valvular dysfunction. Heart valve fibrosis not only affects the normal opening and closing of the valves but also leads to a gradual decline in heart function, severely impacting patients' quality of life and lifespan. From a pathophysiological perspective, heart valve fibrosis is mainly characterized by excessive deposition of extracellular matrix, such as collagen, in the valvular tissue, leading to hardening and thickening of the valvular tissue, and loss of normal elasticity and function. This process typically involves multiple cell types, including valvular interstitial cells, fibroblasts, and inflammatory cells. Currently, there is no effective treatment for heart valve fibrosis, primarily due to the lack of effective in vitro disease research models. Therefore, establishing a new model of heart valve fibrosis is of great significance for understanding the mechanisms related to heart valve fibrosis and for the development of new drugs.

[0003] In recent years, the rapid development of bioprinting technology has provided new possibilities for constructing in vitro disease models. This revolutionary technology integrates the latest achievements in multiple fields such as materials science, biomedical engineering, and tissue engineering. Through bioprinting, the spatial distribution of cells, biomaterials, and bioactive molecules can be precisely controlled, thereby constructing three-dimensional tissue models that more closely resemble the physiological structure and function of the human body. This technology shows enormous application potential in fields such as tissue engineering, regenerative medicine, and drug screening.

[0004] However, there is currently a lack of bioprinted models of heart valve fibrosis that can accurately reflect the three-dimensional structure and intercellular interactions of human tissues and better simulate the effects of the in vivo environment on valve tissue. Summary of the Invention

[0005] The purpose of this invention is to provide a bioprinted model of heart valve fibrosis, its preparation method, and its application. This model has excellent performance and can be used for research on related disease mechanisms and screening of related drugs.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing a bioprinted model of heart valve fibrosis, comprising the following steps:

[0008] A valve model was printed using a hydrogel containing valve cells as the printing material and digital light processing technology.

[0009] The digital light processing technology includes importing design drawings of different grayscale levels into a digital light processing printing device, and then culturing them to obtain bioprinted heart valve fibrosis disease models with different hardness.

[0010] Preferably, the valve cells include valve endothelial cells and / or valve interstitial cells.

[0011] Preferably, the valve cells are obtained through stem cell-induced differentiation.

[0012] Preferably, the stem cells include embryonic stem cells and / or induced pluripotent stem cells.

[0013] Preferably, the concentration of the valve cells in the hydrogel is 10. 5 ~10 7 per mL.

[0014] Preferably, the matrix material of the hydrogel includes one or more of the following: methacrylamide gelatin, polyoxyethylene, methacrylamide hyaluronic acid, polyethylene glycol diacrylate, poloxamer, sodium alginate, chitosan, collagen, fibrinogen, polylactic acid-glycolic acid copolymer, polycaprolactone, silk fibroin, or polyhydroxyethyl methacrylate.

[0015] Preferably, the concentration of the matrix material of the hydrogel is 0.1–20 wt%.

[0016] Preferably, the grayscale range is 10 to 255, and the grayscale corresponds to the intensity of ultraviolet light in the subsequent digital light processing technology;

[0017] And / or, the hardness ranges from 0.1 to 100 kPa;

[0018] And / or, the culture period is 3 to 20 days.

[0019] The present invention also provides a bioprinted model of heart valve fibrosis disease prepared by the above preparation method.

[0020] This invention also provides the application of the above-described bioprinted heart valve fibrosis disease model in any of the following:

[0021] ① Research on the mechanism of cardiac valve fibrosis;

[0022] ② Research and development of drugs for heart valve fibrosis;

[0023] ③ Development of diagnostic technologies for valvular fibrosis;

[0024] ④ Prognostic assessment of valvular fibrosis;

[0025] ⑤ Design of surgical plans for heart valve repair or replacement;

[0026] ⑥ Research and development of medical devices related to heart valve fibrosis;

[0027] ⑦ Research on gene therapy protocols for valvular fibrosis;

[0028] ⑧ Pathological teaching of valvular fibrosis;

[0029] ⑨ Establishment of a high-throughput platform for drug screening in patients with valvular fibrosis;

[0030] ⑩ Immunotherapy research for valvular fibrosis.

[0031] The beneficial effects of this invention are:

[0032] The DLP printing method used in this invention can accurately replicate the layered structure of valves and provides better support for the in vitro construction of valve tissue by optimizing the mechanical properties and biocompatibility of biomaterials. Traditional two-dimensional cell culture and animal models have many limitations in drug screening, such as failing to accurately reflect the three-dimensional structure of human tissues and intercellular interactions, as well as biases in drug efficacy assessment caused by interspecies differences. The three-dimensional cardiac valve fibrosis model provided by this invention can effectively overcome these shortcomings and provide more reliable preclinical data for new drug development.

[0033] This invention applies DLP printing technology to the construction of a model of valvular fibrosis, which not only allows for a better understanding of the disease's pathogenesis but also provides a more precise and efficient platform for high-throughput drug screening. Extracellular matrix stiffness can regulate the degree of fibrosis in various mechanoresponsive cells (fibroblasts, myofibroblasts, and mesenchymal stem cells). By adjusting DLP printing parameters, the degree of cellular fibrosis can be controlled, simulating different levels of fibrosis and better mimicking the effects of the in vivo environment on valvular tissue. Attached Figure Description

[0034] Figure 1 is a grayscale schematic diagram of the printed valve fibrosis model;

[0035] Figure 2 shows a scanning electron microscope image of the hydrogel in the valve fibrosis model;

[0036] Figure 3 shows the fluorescence staining of cell viability in the valve fibrosis model;

[0037] Figure 4 shows the expression of fibrosis markers mRNA in cells in the valve fibrosis model.

[0038] Figure 5 shows the fluorescence staining of fibrosis indicators in cells from different grayscale valve fibrosis models. Detailed Implementation

[0039] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0040] Example

[0041] (1) VIC cells were obtained by inducing differentiation of iPS cells;

[0042] (2) The VIC cells from step (1) were prepared at a concentration of 10 6 Cell-containing printing ink is obtained by mixing cells / ml with 7% GelMA and 0.4% PEO;

[0043] (3) Create a 1920*1080 black background canvas using Photoshop. Create 24 circular images on the canvas and divide them into four groups of six with different gray levels (25, 50, 100, 200). Each circle has 300 pixels, and the primitive diameter is 8mm after DLP projection, as shown in Figure 1.

[0044] (4) Pour the printing material from step (2) into the DLP printing device, import the grayscale image from step (3) into the DLP printing device, and then expose the polymer printing material. The DLP device uses a 0.47-inch DMD chip with a resolution of 1920*1080.

[0045] (5) The cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 7 days to obtain a cardiac valve fibrosis positional disease model that was regulated by matrix stiffness and used for drug screening.

[0046] Experimental Example

[0047] A valve fibrosis model was prepared by DLP printing according to the method described in the example. After lyophilization, it was observed using a scanning electron microscope, and the results are shown in Figure 1. The results show that the hydrogel model prepared by DLP printing has uniform pore size.

[0048] To assess the biocompatibility of the prepared valvular fibrosis model, cells within the model were stained with cell viability and death dyes to observe their survival. Green signals represent live cells, and red signals represent dead cells, as shown in Figure 2. Figure 2 indicates that the valvular fibrosis model has good biocompatibility.

[0049] Different substrates with varying hardness were prepared using printing conditions of different gray levels (high, medium-high, medium, and low) to simulate different degrees of valvular fibrosis. Figure 3 shows the mRNA expression levels of fibrosis markers (α-SMA, Col1A1, and CTGF) extracted from cells in the valvular fibrosis model after 7 days of culture. The mRNA expression levels of each fibrosis marker decreased with decreasing gray level. This result indicates that by adjusting the gray level, this model can accurately simulate different degrees of valvular fibrosis.

[0050] Different substrates with varying hardness were prepared using printing conditions with different gray levels (high, medium-high, medium, and low) to simulate different degrees of valvular fibrosis. Figure 4 shows the immunofluorescence staining analysis of fibrosis markers (α-SMA, red fluorescence) in cells from the valvular fibrosis model after 7 days of culture. The fluorescence intensity of α-SMA decreased with decreasing gray level. This result indicates that by adjusting the gray level, this model can accurately simulate different degrees of valvular fibrosis.

[0051] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a bioprinted model of heart valve fibrosis, characterized in that, Includes the following steps: A valve model was printed using a hydrogel containing valve cells as the printing material and digital light processing technology. The digital light processing technology includes importing design drawings of different grayscale levels into a digital light processing printing device, and then culturing them to obtain bioprinted heart valve fibrosis disease models with different hardness.

2. The preparation method according to claim 1, characterized in that, The valve cells include valve endothelial cells and / or valve interstitial cells.

3. The preparation method according to claim 2, characterized in that, The valve cells were obtained through stem cell-induced differentiation.

4. The preparation method according to claim 3, characterized in that, The stem cells include embryonic stem cells and / or induced pluripotent stem cells.

5. The preparation method according to claim 1, characterized in that, The concentration of the valve cells in the hydrogel is 10. 5 ~10 7 per mL.

6. The preparation method according to claim 1 or 5, characterized in that, The matrix material of the hydrogel includes one or more of the following: methacrylamide gelatin, polyoxyethylene, methacrylamide hyaluronic acid, polyethylene glycol diacrylate, poloxamer, sodium alginate, chitosan, collagen, fibrinogen, polylactic acid-glycolic acid copolymer, polycaprolactone, silk fibroin, or polyhydroxyethyl methacrylate.

7. The production method according to claim 6, characterized by, The concentration of the matrix material of the hydrogel is 0.1–20 wt%.

8. The preparation method according to claim 1, characterized in that, The grayscale range is 10 to 255, and the grayscale corresponds to the intensity of ultraviolet light in the subsequent digital light processing technology. And / or, the hardness ranges from 0.1 to 100 kPa; And / or, the culture period is 3 to 20 days.

9. A bioprinted model of heart valve fibrosis disease prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the bioprinted heart valve fibrosis disease model according to claim 9 in any of the following: ① Research on the mechanism of cardiac valve fibrosis; ② Research and development of drugs for heart valve fibrosis; ③ Development of diagnostic technologies for valvular fibrosis; ④ Prognostic assessment of valvular fibrosis; ⑤ Design of surgical plans for heart valve repair or replacement; ⑥ Research and development of medical devices related to heart valve fibrosis; ⑦ Research on gene therapy protocols for valvular fibrosis; ⑧ Pathological teaching of valvular fibrosis; ⑨ Establishment of a high-throughput platform for drug screening in patients with valvular fibrosis; ⑩ Research on immunotherapy for valvular fibrosis.

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