A method of additive manufacturing of biological soft tissue in a gravity-free suspension

Abstract

The application discloses a kind of gravity-free suspension biological soft tissue additive manufacturing method, belong to biological tissue additive manufacturing technical field.This method includes the following steps: (1) by 3D printing to be made into the three-dimensional morphological structure of target organ or soft tissue blood vessel, and the intensity of the structure is controlled;(2) fixed three-dimensional morphological structure of blood vessel, make it in stable suspended state in gravity-free printing device;(3) based on the type of target organ or soft tissue, corresponding biological material is adhered to three-dimensional morphological structure of blood vessel, and the additive manufacturing of target organ or soft tissue is carried out layer by layer;(4) remove three-dimensional morphological structure of blood vessel, cultivate target organ or soft tissue under gravity-free environment, and make it develop into complete tissue or organ.The application avoids the collapse or separation of biological material and blood vessel structure, solves the technical problems of accurate positioning and printing control under gravity-free environment, and has wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biological tissue additive manufacturing technology, and in particular to a method for additive manufacturing of biological soft tissue under zero gravity suspension. Background Technology

[0002] The application of 3D bioprinting in the medical field is mainly concentrated in organ transplantation. Its goal is to create organs that match the patient's anatomy and tissue characteristics, thereby improving the success rate of transplantation surgery and the patient's quality of life. 3D bioprinting first requires the digital design of the desired biological model or tissue structure, which can be accomplished using computer-aided design software or medical image processing software. After design, biomaterials, such as cells, cell culture media, and scaffold materials, are used as printing raw materials, and these biomaterials are deposited layer by layer into predetermined locations to construct the desired biological structure. The printing process can be achieved through various methods, such as inkjet bioprinting, laser sintering, and gel extrusion. After 3D bioprinting is completed, post-processing can be performed according to actual needs, such as cross-linking, curing, cleaning, and culturing, to promote the binding, maturation, and growth of the biomaterials. 3D bioprinters can customize the manufacture of biological tissues and organs according to each individual's specific needs and anatomical characteristics. This personalized manufacturing capability provides better adaptability and effectiveness for medical treatment and prosthetic implantation.

[0003] For example, Chinese invention patent CN112891633A discloses a method for fabricating a three-dimensional vascularized myocutaneous flap based on bio-3D printing. Utilizing coaxial bio-3D printing, a calcium chloride solution is used as the inner layer, and a mixture of methacrylic anhydride gelatin, sodium alginate, polyethylene glycol diacrylate, and vascular smooth muscle cells is used as the outer bio-ink. The printed scaffold is thoroughly cross-linked through calcium chloride and blue light exposure, ultimately forming a three-in-one perfusion scaffold to establish blood supply. Vascular endothelial cells are perfused within the scaffold to simulate vascular cell components. Fibroblasts, adipose-derived mesenchymal stem cells, and skeletal muscle myoblasts are seeded onto the three layers of the scaffold, respectively, and cultured in a reactor using fibroblast culture medium, adipogenesis induction culture medium, and myoblast culture medium. The three layers of the scaffold are then folded and bonded together using rat tail collagen to form a stacked scaffold. This invention achieves integrated blood perfusion and independent culture, differentiation, and assembly of different cell components. However, the survival rate of vascular endothelial cells and different cell types within the scaffold in this approach is uncertain in practice. The survival rate during 3D printing can be affected by factors such as printing pressure, cross-linking process, and perfusion speed, leading to a decrease in survival rate. Furthermore, the scaffold structure needs to remain stable during folding and assembly to maintain the vascularized structure. Insufficient or unstable cross-linking of the material can cause structural collapse or deformation.

[0004] For example, Chinese invention patent CN116218760A discloses an arterial organ-on-a-chip based on multi-material suspension bio-3D printing and its preparation method. The bio-ink material is derived from decellularized matrix of mammalian soft tissue. Taking porcine decellularized matrix (dECM) as an example, this invention combines ruthenium chloride (Ru) and sodium persulfate (SPS) to formulate a bio-ink material, VLC-dECM, with photo-irradiation crosslinking properties. Water-soluble PF-127 is used as a sacrificial ink material. The process involves sequentially suspending and printing a first layer of VLC-dECM material encapsulating human aortic vascular smooth muscle cells and a second layer of sacrificial material PF-127 in a support bath. After visible light crosslinking, the sacrificial material PF-127 is dissolved and removed using phosphate-buffered saline (PBS), forming a vascular channel. This invention saves manufacturing time; the manufactured arterial organ-on-a-chip can achieve pump-free perfusion under gravity guidance, simplifying the overall structure of the arterial organ-on-a-chip; and the manufactured arterial organ-on-a-chip possesses a good multi-layered arterial biomimetic structure. However, the process of multi-material suspension bio-3D printing requires highly precise control of parameters including temperature, light, printing speed and air pressure. Moreover, the structural stability of arterial organ-on-a-chip depends on the interpenetrating network formed by photocrosslinking and thermo-crosslinking. The materials in different parts are affected by temperature and light to varying degrees, resulting in uneven development of structural stability.

[0005] Because biological soft tissues have a low elastic modulus, printed biological tissues are prone to collapse under the influence of gravity before they have fully developed or before connections have been established between cells. Furthermore, current additive manufacturing methods use high-concentration bio-inks to constrain cells, but excessive external materials significantly hinder intercellular communication, making it difficult to achieve the additive manufacturing of perfect organs.

[0006] Literature (Xingwu Mo # Yanmei Zhang # Zixuan Wang #(Xianhao Zhou, Zhenrui Zhang, Yongcong Fang*, Zilian Fan, Yihan Guo, Ting Zhang*, Zhuo Xiong*. Satellite-based On-orbit Printing of 3D Tumor Models. Advanced Materials, 2023.) This report describes the successful launch and in-orbit testing of a tumor model space 3D printing and culture system developed by the BRE team of the Department of Mechanical Engineering at Tsinghua University. This pioneering work marks a significant step in space 3D printing of tumor models, paving the way for applications of zero-gravity 3D bioprinting. This method utilizes the unique microgravity environment of space to facilitate the construction of precise biomimetic tumor models using 3D bioprinting methods. These models can be used to assess the combined effects of the space environment on tumors, thereby gaining a deeper understanding of the disease's mechanisms and potential treatments. The construction of precise tumor pathology models through space bioprinting is of great significance for space-based tumor research.

[0007] Space printing technology, or zero-gravity printing technology, holds the promise of completely resolving the adverse effects on the processing of biological soft tissues due to their low elastic modulus. However, zero-gravity printing technology is still in the exploratory stage, and several technical problems remain to be solved. The most pressing issue is how to address the difficulty in positioning and precisely controlling biological materials in a zero-gravity environment, which is key to achieving additive manufacturing of suspended biological soft tissues in zero gravity.

[0008] In summary, this paper presents a feasible technical solution for the zero-gravity printing of soft tissue biological organs, which can effectively solve the problems of printing positioning and precise control in a zero-gravity environment, and is of great significance for expanding the application of 3D bioprinting. Summary of the Invention

[0009] In view of the above-mentioned deficiencies of the prior art, in a first aspect of the present invention, a method for additive manufacturing of low-elastic modulus materials in a weightless environment is provided, comprising the following steps:

[0010] (1) Using hydrolyzable or soluble materials as printing raw materials, 3D printing is used to make them into three-dimensional morphological structures of blood vessels in target organs or soft tissues, and the strength of the structure is controlled.

[0011] (2) Fix the three-dimensional morphological structure of the blood vessel so that it is in a stable suspended state in the zero-gravity printing device;

[0012] (3) Based on the type of target organ or soft tissue, the corresponding biomaterials are extruded and adhered to the three-dimensional morphological structure of blood vessels, and additive manufacturing of target organ or soft tissue is carried out layer by layer.

[0013] (4) After the cells to be added are cultured and connected with other cells, the three-dimensional morphological structure of blood vessels is hydrolyzed or dissolved, and the target organ or soft tissue is cultured in a weightless environment so that it develops into a complete tissue or organ.

[0014] Preferably, in step (1), the type of printing material includes at least one of polylactic acid, polycaprolactone, and starch-based materials.

[0015] The printed three-dimensional morphological structure of the blood vessel needs to have good strength and toughness to meet the requirements of biological applications while taking into account the process stability requirements in a zero-gravity environment.

[0016] Preferably, in step (1), the strength indicators include tensile strength, elongation at break, elastic modulus, and flexural modulus.

[0017] More preferably, the tensile strength is controlled within the range of 40-60 MPa.

[0018] More preferably, the elongation at break is controlled within the range of 4%-10%.

[0019] More preferably, the elastic modulus is controlled within the range of 300-400 MPa.

[0020] More preferably, the bending modulus is controlled within the range of 10-150 MPa.

[0021] Preferably, in step (2), the main blood vessel end of the three-dimensional morphological structure of the blood vessel is fixed to the top of the zero-gravity printing device, so that the part of the three-dimensional morphological structure of the blood vessel that is not connected to the fixed end is suspended in the air.

[0022] Preferably, in step (4), during the cultivation process, a corresponding nutrient solution is supplied according to the cell growth characteristics of the target organ or soft tissue; the nutrient solution contains nutrients including sugars, inorganic salts, growth factors, and vitamins.

[0023] Based on the above technical solutions, the inventors' design concept is to model the blood vessel distribution and structure of real soft tissues or organs, and then use conventional methods in the field to 3D print a vascular structure model (three-dimensional morphological structure of blood vessels) of the target soft tissue or organ. The equipment used to print the vascular structure model of the desired tissue or organ is versatile and can be any existing additive manufacturing equipment, including extrusion printing equipment, photopolymerization printing equipment, and laser powder bed printing equipment. The printing raw material is a hydrolyzable or rapidly degradable material that rapidly hydrolyzes or degrades during the cultivation process after the entire tissue or organ is printed, forming a duct structure.

[0024] The printed vascular structure is placed in a zero-gravity printing device, with the main vessel end fixed to the device, allowing the portions of the vascular structure not connected to the fixed end to suspend in mid-air. The inventors considered the overall suspension of the vascular structure during fixation, ensuring more of the vessel remains suspended, which facilitates the precise printing of subsequent cells and other biomaterials. After the printed vascular structure is fixed, different cells or biomaterials are extruded using different extrusion needles, performing additive manufacturing along the vessel to adhere them to the structure. Due to the zero-gravity environment, the cells and other biomaterials adhere to the vascular structure without collapsing or separating. This layered printing based on vascular structures effectively provides support for zero-gravity bioprinting, making positioning and precise control during the printing process easier. Considering the zero-gravity preparation environment, extrusion is suitable for printing; inkjet printing may result in suspended biomaterial particles, lower utilization rates, and is detrimental to tissue culture.

[0025] Once the complete shape of a biological soft tissue or organ is printed out, it can be directly cultured in a zero-gravity environment until the soft tissue or organ is fully developed and forms a biologically functional organ or tissue.

[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0027] This invention provides a method for additive manufacturing of biological soft tissue under zero gravity suspension, which avoids the collapse or separation of biological materials and vascular structures, and solves the technical problems of precise positioning and printing control of biological soft tissues or organs in a zero-gravity environment. It can efficiently prepare organs or soft tissues and has broad application prospects. Attached Figure Description

[0028] Figure 1 This is a process flow diagram of the zero-gravity suspended biological soft tissue additive manufacturing method of Example 1;

[0029] Figure 2 This is a process flow diagram of the zero-gravity suspension biological soft tissue additive manufacturing method in Example 2. Detailed Implementation

[0030] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0031] Example 1

[0032] This embodiment demonstrates the process for preparing a lung based on a gravity-free suspension bio-soft tissue additive manufacturing method, as follows: Figure 1 As shown, the steps are as follows:

[0033] (1) Using hydrolyzable or soluble starch-based materials as printing raw materials, the distribution and structure of blood vessels in real lung tissue are modeled, and the three-dimensional morphological structure of blood vessels in lung organs is printed using traditional 3D printing equipment; the strength development of the structure is controlled so that the tensile strength is controlled within the range of 40-60MPa, the elongation at break is controlled within the range of 4%-10%, the elastic modulus is controlled within the range of 300-400MPa, and the flexural modulus is controlled within the range of 10-150MPa, so that the printed blood vessels have good strength and toughness and can be used for blood transport.

[0034] (2) Fix the lung main blood vessel end of the three-dimensional morphological structure of lung tissue blood vessels to the top of the zero-gravity printing device so that most of the lung blood vessel structure is suspended in the air. When fixing, the overall suspension of the lung blood vessels needs to be fully considered so that more lung blood vessels are suspended in the air, which is conducive to the subsequent printing of different lung cells and other biomaterials.

[0035] (3) After the three-dimensional morphological structure of lung tissue blood vessels is fixed, different lung cells and biomaterials are extruded using different extrusion needles and additive manufacturing is carried out along the lung blood vessel structure. The cells and other biomaterials are adhered to the blood vessel structure. Due to the weightless environment, the lung cells and other biomaterials can adhere to the blood vessel structure without collapsing or separating. This layered printing based on the lung blood vessel structure can effectively provide support for weightless bioprinting, thus making it easier to position and control precisely during the printing process.

[0036] (4) After the cells have been cultured and established connections with other cells, the three-dimensional morphological structure of the blood vessels is hydrolyzed to form formal blood vessels. Finally, the cells are cultured in a weightless environment, during which time the corresponding nutrient solution is supplied according to the cell growth characteristics until the lung tissue and organs are fully developed and a lung organ with biological functions is formed.

[0037] Example 2

[0038] This embodiment demonstrates the process of fabricating a heart based on a gravity-free suspension bio-soft tissue additive manufacturing method, as follows: Figure 2 As shown, the steps are as follows:

[0039] (1) Using hydrolyzable or soluble starch-based materials as printing raw materials, the distribution and structure of blood vessels in real heart tissues and organs are modeled, and the three-dimensional morphological structure of blood vessels in heart organs is printed using traditional 3D printing equipment; the strength development of the structure is controlled so that the tensile strength is controlled within the range of 40-60MPa, the elongation at break is controlled within the range of 4%-10%, the elastic modulus is controlled within the range of 300-400MPa, and the flexural modulus is controlled within the range of 10-150MPa, so that the printed blood vessels have good strength and toughness and can be used for blood transport;

[0040] (2) Fix the main blood vessel end of the three-dimensional morphological structure of the heart tissue blood vessels to the top of the zero-gravity printing device so that most of the heart blood vessel structure is suspended in the air. When fixing, the overall suspension of the heart blood vessels needs to be fully considered so that more heart blood vessels are suspended in the air, which is conducive to the subsequent printing of different tissue cells and other biomaterials from different parts of the heart.

[0041] (3) After the three-dimensional morphological structure of the heart tissue blood vessels is fixed, different extrusion needles are used to extrude cells and biomaterials from different parts of the heart. Additive manufacturing is carried out along the heart blood vessel structure, and the cells and other biomaterials are adhered to the blood vessel structure. Due to the weightless environment, the heart cells and other biomaterials can adhere to the blood vessel structure without collapsing or separating. This layered printing based on the heart blood vessel structure can effectively provide support for weightless bioprinting, thus making it easier to position and control precisely during the printing process.

[0042] (4) After the cells have been cultured and established connections with other cells, the three-dimensional morphological structure of the blood vessels is hydrolyzed to form formal blood vessels. Finally, the cells are cultured in a weightless environment, during which time the appropriate nutrient solution is supplied according to the cell growth characteristics until the heart tissue and organs are fully developed and a biologically functional heart organ is formed.

[0043] The zero-gravity suspension biological soft tissue additive manufacturing method of the present invention avoids the collapse or separation of biological materials and vascular structures, solves the technical problems of precise positioning and printing control of biological soft tissues or organs in a zero-gravity environment, and can efficiently prepare organs or soft tissues, with broad application prospects.

[0044] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A method for additive manufacturing of weightless suspended biological soft tissue, characterized in that, Includes the following steps: (1) Using hydrolyzable or soluble materials as printing raw materials, 3D printing is used to make them into three-dimensional morphological structures of blood vessels in target organs or soft tissues, and the strength of the structure is controlled. (2) Fix the three-dimensional morphological structure of the blood vessel so that it is in a stable suspended state in the zero-gravity printing device; (3) Based on the type of target organ or soft tissue, the corresponding biomaterials are extruded and adhered to the three-dimensional morphological structure of blood vessels, and additive manufacturing of target organ or soft tissue is carried out layer by layer. (4) After the cells to be added are cultured and connected with other cells, the three-dimensional morphological structure of blood vessels is hydrolyzed or dissolved, and the target organ or soft tissue is cultured in a gravity-free environment so that it develops into a complete tissue or organ.

2. The method according to claim 1, characterized in that: In step (1), the type of printing raw material includes at least one of polylactic acid, polycaprolactone, and starch-based materials.

3. The method according to claim 1, characterized in that: In step (1), the strength indicators include tensile strength, elongation at break, elastic modulus, and flexural modulus.

4. The method according to claim 3, characterized in that: The tensile strength is controlled within the range of 40-60 MPa.

5. The method according to claim 3, characterized in that: The controlled range of the elongation at break is 4%-10%.

6. The method according to claim 3, characterized in that: The elastic modulus is controlled within the range of 300-400 MPa.

7. The method according to claim 3, characterized in that: The bending modulus is controlled within the range of 10-150 MPa.

8. The method according to claim 1, characterized in that: In step (2), the main blood vessel end of the three-dimensional morphological structure of the blood vessel is fixed to the top of the zero-gravity printing device, so that the part of the three-dimensional morphological structure of the blood vessel that is not connected to the fixed end is suspended in the air.

9. The method according to claim 1, characterized in that: In step (4), during the cultivation process, a corresponding nutrient solution is supplied according to the cell growth characteristics of the target organ or soft tissue; the nutrient solution contains nutrients including sugars, inorganic salts, growth factors, and vitamins.

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