A micro-nano fiber composite cell / hydrogel muscle tissue 3D printing method

Abstract

A kind of muscle tissue 3D printing method of micro-nano fiber composite cell / hydrogel, the accurate manufacture of large volume active muscle tissue and the directional bundle growth of muscle cell are realized by multi-material printing technology;Biocompatible material is made into micro-nano fiber scaffold by electrostatic printing technology or micro-nano 3D printing technology, which provides physical contact structure induction for cells, guides the growth and arrangement of cells;By mixing cells with various hydrogels, cell-carrying biological ink with suitable concentration is formed, and then through 3D printing technology, the spatial structure and composition of cell / hydrogel in micro-nano fiber scaffold are accurately controlled, forming micro-nano fiber / hydrogel / cell composite structure;The composite structure is pre-cultured in vitro, and the differentiation, development and maturation of muscle tissue are further promoted by using dynamic bioreactor and biochemical component stimulation;This method is suitable for various cell types and biomaterials, and provides a technical basis for the stability and functional reconstruction of large volume muscle tissue.

Description

Technical Field

[0001] This invention relates to the field of 3D printing and tissue engineering technology, specifically to a method for 3D printing muscle tissue from micro / nanofiber composite cells / hydrogels. Background Technology

[0002] Skeletal muscle is an important component of the human body, playing a vital role in maintaining posture, movement, respiration, and blood circulation. Skeletal muscle loss caused by accidents, injuries, or tumor surgery with resection is quite common. While the body's stem cells have some ability to repair muscle damage, when more than 20% of a skeletal muscle is lost, it becomes difficult to repair. Skeletal muscle that cannot repair itself undergoes atrophy, forming scar tissue at the wound site, ultimately leading to muscle weakness and wound depression. More severe muscle loss can result in larger areas of tissue necrosis, amputation, and disability.

[0003] Currently, the most widely used treatment for muscle defects in clinical practice is autologous muscle tissue transplantation. However, cases of transplanted muscle necrosis, inability to reconnect with wound tissue, loss of contractile function, and subsequent atrophy often occur. Therefore, autologous muscle tissue transplantation is a method that sacrifices healthy tissue and carries a certain risk of failure, and it cannot perfectly solve the problem of muscle defect treatment.

[0004] Muscle tissue engineering is a promising alternative to autologous transplantation for treating skeletal muscle defects. It involves obtaining a small amount of living skeletal muscle tissue, isolating skeletal muscle stem cells from the tissue block using specific methods, culturing and expanding them in vitro, and then mixing the expanded cells with biocompatible, biodegradable, and absorbable biomaterials in a specific ratio. This allows the cells to adhere to the biomaterials, forming a muscle tissue engineering scaffold. This scaffold is then implanted into the skeletal muscle defect site. As the biomaterials are gradually degraded and absorbed in vivo, the implanted skeletal muscle stem cells proliferate, grow in a directed manner, differentiate, and fuse, ultimately forming mature muscle fibers. Simultaneously, surrounding blood vessels gradually grow into the scaffold, and motor neuron axons penetrate the mature muscle fibers, establishing neuromuscular connections. This achieves the goal of repairing trauma and rebuilding muscle function. The skeletal muscle scaffold not only provides a favorable biological environment for the directed growth, nutrient acquisition, and metabolism of skeletal muscle stem cells but also provides space for the growth and repair of other tissues. The development of skeletal muscle tissue engineering provides a technological means for skeletal muscle tissue regeneration, which will change the traditional surgical treatment model of "repairing trauma with trauma."

[0005] Currently, artificial skeletal muscle tissue mainly employs several methods, including electrospinning, melt direct writing, hydrogel infusion molding, and hydrogel extrusion 3D printing. Among these, electrospinning and melt direct writing can induce muscle cell growth through cell-scale structures, but due to the decrease in electric field strength with increasing height, it is difficult to form large-volume fiber scaffolds from a molding perspective, thus making it impossible to construct three-dimensional muscle tissue with clinical dimensions (Materials Science & Engineering C 116(2020)111070; International Journal of Pharmaceutics 606(2021)120841). Hydrogel casting and extrusion 3D printing technology (Biocybernetics and Biomedical Engineering 38(1)(2018)158-169) can simulate the fine directional structure of muscle, but it suffers from insufficient mechanical strength and collapses due to contraction during the long-term repair of large-volume muscle defects. Summary of the Invention

[0006] To overcome the shortcomings of existing methods, the present invention aims to provide a 3D printing method for muscle tissue using micro / nanofiber composite cells / hydrogels. This method can precisely control the ratio and spatial arrangement of various cells with submicron or nanoscale fibers. At the same time, the fine fibers provide structural induction and mechanical support for muscle tissue, guiding and constraining the growth and arrangement of cells during in vitro culture. This enables the manufacture of large-volume active muscle tissue and the directional bundled growth of muscle cells, providing long-term and effective mechanical support for the repair of large-volume muscle defects.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] A method for 3D printing muscle tissue using micro / nanofiber composite cells / hydrogels includes the following steps:

[0009] Step 1: Prepare the cell-loaded bio-ink. The cell-loaded bio-ink is composed of a mixture of cells and hydrogel, with a cell concentration of 1×10⁻⁶. 5 -5×10 7 Cells / ml, used for perfusion or printing of viable cells;

[0010] Step 2: Use electrostatic printing or micro / nano 3D printing technology to manufacture biocompatible materials into micro / nano fiber scaffolds, which serve as structural induction factors for physical contact, guiding the growth, attachment, and arrangement of cells; then, inject or composite 3D print cell-loaded bio-ink on demand into specific locations within the micro / nano fiber scaffold to obtain a micro / nano fiber / hydrogel / cell composite structure with controllable spatial structure and composition distribution.

[0011] Step 3: Pre-culture the micro / nanofiber / hydrogel / cell composite structure in vitro, so that the cells grow, proliferate and arrange as needed under the guidance and constraint of the micro / nanofiber structure, to achieve a composite tissue with cell bundles.

[0012] The composite tissue with cell bundles is placed inside a dynamic bioreactor for further culture, supplemented by culture medium and / or physical stimulation conditions such as force, electricity, and magnetism, to continue to guide the differentiation, development and maturation of the cultured meat tissue.

[0013] Dynamic bioreactors also contain biochemical stimuli, and the addition of culture medium containing biochemical factors varies depending on the type of cultured tissue and its growth and differentiation stage.

[0014] The stimulation conditions applied to the cultured tissue include stretching, shearing, compression, vibration, optical, acoustic and electromagnetic stimuli. The stimuli are applied continuously or intermittently, and the frequency, amplitude and duration of application are related to the texture of the cultured tissue. The electromagnetic field of electromagnetic stimulation includes constant, pulsed and alternating electric and magnetic fields. Compression includes static compression and dynamic compression.

[0015] In step 1, the hydrogel in the cell-carrying bio-ink is composed of collagen, fibrin, gelatin, Matrigel, hyaluronic acid, sodium alginate, polyvinyl alcohol, and modified hydrogels thereof; the cells are human or animal muscle cells, vascular endothelial cells, adipocytes, liver cells, and mesenchymal stem cells.

[0016] The biocompatible materials in step 2 include polycaprolactone (PCL), polylactic acid, chitosan, silk fibroin, etc.

[0017] In step 2, the combination of micro / nanofiber scaffold printing and cell-carrying bio-ink printing processes is achieved through automated continuous printing or partially / completely independent printing of micro / nanofiber scaffolds and cell-carrying hydrogel structures, followed by assembly.

[0018] The 3D printing methods for micro / nano fiber scaffolds in step 2 include electrostatic printing, direct-write 3D printing, micro / nano 3D printing, two-photon 3D printing, and immersion volume printing; the fiber morphology includes circular and elliptical shapes, such as fibers with special concave-convex morphologies and heterogeneous components printed using coaxial needles or other irregularly shaped needles; the 3D structure of the micro / nano fiber scaffold is precisely customized according to specific tissue types and cell guidance methods.

[0019] In step 2, the 3D printing methods for cell-borne bio-ink include electrostatic printing, extrusion direct writing 3D printing, photopolymerization 3D printing, and immersion volume printing. The cross-linking method of the hydrogel can be freely selected, including temperature cross-linking, pH cross-linking, ionic cross-linking, enzyme cross-linking, and photopolymerization cross-linking.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] 1) Compared with the prior art, the present invention utilizes multi-material printing technology to realize the manufacturing of composite muscle tissue with large thickness and large volume, wherein the composition, structure and distribution of active cells and micro-nano fibers can be precisely adjusted on demand at the micron scale;

[0022] 2) Under the guidance of hydrogel and micro / nanofiber scaffolds, cells can achieve growth patterns that mimic natural tissues. During growth, the pores generated by hydrogel and cell contraction can ensure the supply of nutrients. Vascular endothelial cells can also promote the vascularization of large-volume muscle tissue, which is beneficial to the in vitro survival and growth of large-volume muscle.

[0023] 3) The printed cultured tissues grow, differentiate, and mature under structural constraints, in vitro force / electric / magnetic stimulation, and biochemical stimulation, and the maturity of the cultured tissues is further regulated during the culture process;

[0024] 4) The method of this invention is not limited to artificial muscles. Different biomimetic structures can be designed and manufactured according to actual needs, and different cells and fibers can be printed to extend its application to the cultivation of different species and different active tissues and organs. Attached Figure Description

[0025] Figure 1 This is a flowchart of the method of the present invention.

[0026] Figure 2 This is a schematic diagram of the micro / nanofiber composite cell / hydrogel 3D printing equipment of Embodiment 1 of the present invention.

[0027] Figure 3 This is a physical image of the 20mm high hydrogel fiber composite structure of Embodiment 1 of the present invention.

[0028] Figure 4 The images show a physical photograph of the large-sized active muscle tissue formed by cell / hydrogel perfusion inside a microfiber scaffold in Example 2 of the present invention, and a microscope image showing the cells gradually orienting and bundling over time.

[0029] Figure 5 This is a diagram showing the bundled growth morphology of H9C2 cardiomyocytes, C2C12 myoblasts, NHDF fibroblasts, and MSCs mesenchymal stem cells within a printed micro / nanofiber scaffold in Example 2 of the present invention. Detailed Implementation

[0030] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Example 1: As Figure 1 As shown, a method for 3D printing muscle tissue from micro / nanofiber composite cells / hydrogels includes the following steps:

[0032] Step 1: Prepare cell-loaded bio-ink. The cell-loaded bio-ink consists of a suspension of skeletal muscle cells and RGD-sodium alginate, with a cell concentration of 5 × 10⁻⁶ cells / mL. 7 Cells / ml, used for printing muscle cells;

[0033] Step 2: Use electrostatic printing technology to fabricate biocompatible materials (polycaprolactone / PCL) into micro / nanofiber scaffolds, which serve as structural induction factors for physical contact, guiding the growth, attachment, and arrangement of cells; then, inject or controllably print cell-loaded bio-ink onto specific locations on the micro / nanofiber scaffolds to obtain a micro / nanofiber / hydrogel / cell composite structure with controllable spatial structure and composition.

[0034] Reference Figure 2 This embodiment employs a micro / nanofiber composite cell / hydrogel 3D printing device, including a multi-material printing nozzle, a multi-nozzle precision flow control module, a high-voltage power supply module, an environmental control module, and a high-precision three-dimensional moving platform. It prints biocompatible micro / nanofiber scaffolds using a molten electrostatic printing nozzle, and prints muscle cells and matrix cells using a cell / hydrogel printing nozzle. Through the switching of multiple nozzles and the coordinated coupling of the high-precision three-dimensional moving platform, 3D printing of micro / nanofiber / hydrogel / cell composite structures is achieved within a constant temperature and humidity clean chamber. Figure 3 As shown, the height of the micro / nanofiber / hydrogel / cell composite structure can reach more than 20 mm, breaking through the height limit of micro / nanofiber scaffolds in existing research (10 mm, Advanced Materials Technologies 7 (2021) 2101197);

[0035] Step 3: Culture the micro / nanofiber / hydrogel / cell composite structure in vitro, allowing cells to grow, proliferate, and arrange as needed under the guidance and constraint of the micro / nanofiber structure, thus achieving a composite tissue with cell bundles.

[0036] Example 2: Figure 1 As shown, a method for 3D printing muscle tissue from micro / nanofiber composite cells / hydrogels includes the following steps:

[0037] Step 1: Prepare cell-loaded bio-ink. The cell-loaded bio-ink consists of a suspension of skeletal muscle cells and RGD-sodium alginate, with a cell concentration of 1×10⁻⁶. 5 Cells / ml, used for printing muscle cells;

[0038] Step 2: Use electrostatic printing technology to fabricate biocompatible materials into micro / nanofiber scaffolds, which serve as structural induction factors for physical contact, guiding the growth, attachment, and arrangement of cells; then, inject or controllably print cell-loaded bio-ink onto specific locations on the micro / nanofiber scaffolds to obtain a micro / nanofiber / hydrogel / cell composite structure with controllable spatial structure and composition.

[0039] Reference Figure 2 This embodiment employs a micro / nanofiber composite cell / hydrogel 3D printing device, including a multi-material printing nozzle, a multi-nozzle precision flow control module, a high-voltage power supply module, an environmental control module, and a high-precision three-dimensional moving platform. The device prints biocompatible micro / nanofiber scaffolds using an electrostatic printing nozzle, and prints muscle cells and matrix cells using a hydrogel printing nozzle. Through the switching of multiple nozzles and the coordinated coupling of the high-precision three-dimensional moving platform, the 3D printing of micro / nanofiber / hydrogel / cell composite structures is achieved.

[0040] Step 3: Culture the micro / nanofiber / hydrogel / cell composite structure in vitro, so that the cells grow, proliferate and arrange as needed under the guidance and constraint of the micro / nanofiber structure, to achieve a composite tissue with cell bundles;

[0041] In this embodiment, cells grow and proliferate in a direction guided by the fibrous scaffold, forming muscle tissue with muscle bundles, such as... Figure 4 As shown; this method is applicable to various cell types, allowing for the simultaneous bundled growth of multiple cells, such as H9C2 cardiomyocytes, C2C12 myoblasts, NHDF fibroblasts, and MSCs mesenchymal stem cells, all of which can achieve bundled growth, meeting the needs of constructing different cell types, such as... Figure 5 As shown;

[0042] Step 4: Place the composite tissue with cell bundles into the dynamic bioreactor for further culture, and continue to guide the differentiation, development and maturation of the cultured meat tissue with the help of culture medium or mechanical, electrical and magnetic stimulation conditions;

[0043] The culture medium containing biochemical factors in the dynamic bioreactor varies depending on the type and growth and differentiation stage of the cultured tissue; the excitation conditions applied to the cultured tissue include stretching, shearing, compression, vibration, optical, acoustic and electromagnetic stimulation factors. The stimulation factors are applied continuously or intermittently, and the frequency, amplitude and duration of action are related to the texture of the cultured tissue. The electromagnetic field of electromagnetic stimulation includes constant, pulsed and alternating electric and magnetic fields, and compression includes static compression and dynamic compression.

[0044] Step 4.1: Activate the biochemical stimulation unit to deliver differentiation culture medium into the culture chamber to promote the growth, development and maturation of the printed tissue;

[0045] Step 4.2: Activate the electromagnetic excitation unit to enhance cell proliferation and extracellular matrix formation through excitation. Fluid shear and stretch stimulation can regulate the distribution of cytoskeleton fibers. Parameter settings include 1-10% tensile strain and 0.1-5Hz frequency. Stretch stimulation is applied for 12 hours per day.

Claims

1. A method for 3D printing of muscle tissue using micro- and nanofiber composite cell / hydrogel, characterized by, The method comprises the following steps: Step 1: Configure cell ink, cell-laden bioink is composed of cells and hydrogel mixed, cell concentration is 1 x 10 5 -5 x 10 7 ml, for perfusion or printing of active cells; Step 2: The biocompatible material is manufactured into a micro-nano fiber scaffold by using electrostatic printing technology or micro-nano 3D printing technology, as a physical contact structural inducer, to guide the growth, adhesion and arrangement of cells; the cell-laden bio-ink is perfused or 3D printed on a specific position in the micro-nano fiber scaffold as needed, to obtain a micro-nano fiber / hydrogel / cell composite structure with controllable spatial structure and component distribution; The composite of the micro-nano fiber scaffold printing and the cell-laden bio-ink printing process is automatically and continuously carried out or partially / fully independently printed to form the micro-nano fiber scaffold and the cell-laden hydrogel structure, and then assembled to realize the composite tissue with cell bundles; Step 3: The micro-nano fiber / hydrogel / cell composite structure is pre-cultured in vitro, so that the cells grow, proliferate and arrange as needed under the guidance and constraint of the micro-nano fiber structure, to realize the composite tissue with cell bundles; The composite tissue with cell bundles is placed in a dynamic bioreactor for further culture, assisted by a culture solution or force, electric, magnetic excitation conditions, to continue to guide the differentiation, development and maturation of the cultivated meat tissue; The culture solution containing biochemical factors in the dynamic bioreactor is different for different types and growth and differentiation stages of the cultivated tissue; the excitation conditions applied to the cultivated tissue include stretching, shearing, compression, vibration, optical, acoustic and electromagnetic stimulation factors, the excitation factors are applied continuously or intermittently, the frequency, amplitude and action time are related to the texture of the cultivated tissue, and the electromagnetic field of the electromagnetic stimulation includes constant, pulse and alternating electric field and magnetic field, and the compression includes static compression and dynamic compression.

2. The method of claim 1, wherein: The hydrogel in the cell-laden bio-ink in step 1 is collagen, fibrin, gelatin, Matrigel, hyaluronic acid, sodium alginate, polyvinyl alcohol and modified hydrogel thereof; the cells are muscle cells, vascular endothelial cells, adipocytes, liver cells and mesenchymal stem cells of human or animals.

3. The method of claim 1, wherein: The biocompatible material in step 2 includes polycaprolactone, polylactic acid, chitosan and silk fibroin.

4. The method of claim 1, wherein: The 3D printing method of the micro-nano fiber scaffold in step 2 includes electrostatic printing, direct writing 3D printing, micro-nano 3D printing, two-photon 3D printing and immersion volume printing; the fiber form includes a circle and an ellipse, and the 3D structure of the micro-nano fiber scaffold is accurately customized according to specific tissue types and cell guidance modes.

5. The method of claim 1, wherein: The 3D printing method of the cell-laden bio-ink in step 2 includes electrostatic printing, extrusion direct writing 3D printing, photocuring 3D printing and immersion volume printing, which is freely selected according to the crosslinking mode of the hydrogel, including temperature crosslinking, pH crosslinking, ion crosslinking, enzyme crosslinking and photocuring crosslinking.

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