Magnetic-mechanical force responsive tissue engineering trachea and preparation method thereof

Abstract

The invention relates to the field of tissue engineering, and particularly provides a magnetic-mechanical force responsive tissue engineering trachea as well as a preparation method and application thereof. According to the magnetic-mechanical force responsive tissue engineering trachea and the preparation method thereof, the magnetic nanoparticles and the cartilage cells are combined, the magnetic-mechanical force responsive tissue engineering trachea is constructed through a 3D printing technology, and high-strength cartilage is formed as a mechanical skeleton of the tissue engineering trachea through remote non-contact mechanical stimulation on the cartilage cells. According to the tissue engineering trachea disclosed by the invention, the mechanical property of cartilage is superior to that of a pure cartilage cell group without magnetic-mechanical stimulation under magnetic-mechanical stimulation.

Description

Technical Field

[0001] The invention relates to the field of tissue engineering, in particular to a magnetic-mechanical force responsive tissue engineering trachea and a preparation method thereof. Background Art

[0002] Airway defects caused by various pathological or clinical conditions, especially long-segment tracheal defects, cannot currently be treated by end-to-end anastomosis of the trachea. At this time, a suitable tracheal substitute is needed to achieve airway reconstruction and regeneration. In recent years, tissue engineering has shown great potential in tracheal replacement and regeneration and has made some breakthroughs. For example, in situ repair of tracheal defects can be achieved by cell implantation and induction of cartilage formation in vivo. Cartilage tissue can provide mechanical strength to prevent collapse. After tissue-engineered trachea transplantation, tracheal hoses with good performance can serve as mechanical support to maintain airway patency; on the other hand, they are not easily degraded in the airway inflammatory environment, which helps the regeneration of tissue structures such as airway epithelium and blood vessels, thereby ensuring the long-term function of tissue-engineered trachea in the airway. Therefore, constructing tissue-engineered cartilage with performance similar to that of natural trachea is the key to achieving airway defect reconstruction and regeneration.

[0003] Under physiological conditions, chondrocytes can sense the mechanical properties of their microenvironment through a series of mechanomechanically sensitive receptors and channels, and affect their synthesis and catabolism by activating complex downstream signaling networks. For example, in articular cartilage, chondrocytes respond to the physical properties of the cartilage extracellular matrix (ECM) and the mechanical forces applied to them during joint loading, and regulate cartilage tissue-related physiological and pathological processes. However, common mechanical stimulation methods such as compression and stretching are not easy to apply in animal experiments, and the microenvironment mechanical properties that rely on material characterization lack spatiotemporal regulation, which limits the scope and prospects of mechanical stimulation in tissue engineering cartilage.

[0004] In recent years, with the rapid development of nanoscience and technology, magnetic nanoparticles have been widely used in the biomedical field due to their unique magnetic properties and good biocompatibility.

[0005] However, there are no reports on a magnetic-mechanical responsive tissue engineered trachea and its preparation method. Summary of the invention

[0006] The technical problem to be solved by the present invention is that the common mechanical stimulation methods in the prior art are not easy to apply in animal experiments and the microenvironment mechanical properties that rely on material characterization lack spatiotemporal control, which limits the application of mechanical stimulation in tissue engineering cartilage. The present invention combines magnetic nanoparticles with chondrocytes and constructs a magnetic-mechanical force responsive tissue engineering trachea through 3D printing technology. When incubated with cells, magnetic nanomaterials can enter the cells through cell endocytosis and generate mechanical movement under the action of an external dynamic magnetic field, thereby generating mechanical force on the intracellular structure. Subsequently, when the cells are subjected to external forces at the piconewton level, the mechanosensitive channels are activated to regulate their related physiological processes.

[0007] The first aspect of the present invention provides a method for preparing a magnetic-mechanical force responsive tissue engineering trachea, comprising the following steps:

[0008] (A) First, the photoinitiator phenyl-2,4,6-trimethylbenzoyl lithium phosphite (LAP) was dissolved in PBS solution to prepare a 0.25% (w / v) initiator standard solution. Then, methacryloyl gelatin (GelMA) was dissolved in the initiator standard solution and heated in a water bath until it was completely dissolved to form a 10% (w / v) methacryloyl gelatin solution. After the temperature dropped to room temperature, the chondrocyte suspension was incubated at 1×10 8 / mL concentration was mixed with GelMA solution, and then magnetic nanoparticles were added into the mixed system at a concentration of 12.5μg / mL;

[0009] Furthermore, the magnetic nanomaterial is Fe3O4 nanoparticles with an average hydrodynamic diameter of about 200nm.

[0010] Furthermore, the magnetic nanoparticles are directly immersed in 75% ethanol for 30 minutes before use; and irradiated with ultraviolet light for 30 minutes to be fully sterilized.

[0011] (B) The mixed system prepared in the above steps is printed into tissue-engineered tracheal cartilage rings by a 3D bioprinter under 400-408nm ultraviolet light irradiation; multiple tracheal cartilage rings are stacked layer by layer to form a tissue-engineered trachea with a structure similar to that of a natural gas tube.

[0012] Furthermore, the inner and outer diameters of the tissue engineering tracheal cartilage ring are determined according to the inner and outer diameters of the trachea reconstructed by CT scanning of experimental animals. The printer nozzle movement speed is 10 mm / s, the printing pressure is 0.18 MPa, the temperature is 25°C, and the printing parameters vary depending on the printer model.

[0013] Furthermore, the method for preparing the chondrocyte suspension comprises the following steps:

[0014] The cartilage was minced into 1 mm × 1 mm pieces, and 15% type II collagenase was added. The cells were digested at 37°C for 24 h, with several shakes and blows. The cells were filtered through a 0.22 μm sterile filter, centrifuged, washed, resuspended, and counted. The cells were counted at 1.0 × 10 4 / cm 2 Cells were seeded at a density of 10 cm 2 The cells were cultured in a culture dish with cartilage proliferation medium containing serum and antibiotics; the culture was carried out at 37°C, 5% CO2, and saturated humidity. The medium was changed for the first time on the third day, and then replaced with fresh cartilage proliferation medium every other day; when the cells basically covered the culture dish, about 1.5 mL of trypsin digestion solution was added, and after the cell cytoplasm shrank and the morphology became round under the microscope, DMEM culture medium (containing serum) was added to terminate the digestion, the cells were collected, resuspended after centrifugation, and then 1.0×10 4 / cm 2 The cells were seeded at a new 10 cm 2 The culture dish is prepared, and the above steps are repeated. When the cells are expanded to the second generation, the cells are cultured for 3-5 days until the cells fill the culture dish, and the cells are collected to obtain the chondrocyte suspension.

[0015] Furthermore, the cartilage is rabbit ear cartilage, which is obtained by removing the rabbit ears after anesthesia or killing the rabbit, peeling off the rabbit ear skin, and removing the fibrous membrane around the cartilage tissue.

[0016] The second aspect of the present invention provides a magnetic-mechanical force responsive tissue engineering trachea, which is prepared by the preparation method as described above.

[0017] A third aspect of the present invention provides a use of the magnetic-mechanical force responsive tissue engineered trachea as described above in the preparation of a product for airway defect reconstruction and regeneration.

[0018] After the magnetic-mechanical force responsive tissue engineering trachea of ​​the present invention is embedded in a rabbit, a magnetic field is applied to the experimental rabbit for treatment (the magnetic induction intensity is 260mT, the frequency is 1Hz, the treatment time is 20min / d, and the magnetic field type is a rotating magnetic field). The chondrocytes that have endocytosed the magnetic nanoparticles respond to the in vitro rotating magnetic field and activate the intracellular mechanical signaling pathway, thereby enhancing the chondrogenesis process of the chondrocytes.

[0019] When the present invention is used, it is embedded in the neck muscles around the trachea for 6 weeks of prevascularization. During this period, a rotating magnetic field (magnetic induction intensity of 260mT, frequency of 1Hz, treatment time of 20min / d) is applied in vitro to achieve magnetic-mechanical force stimulation of chondrocytes that have endocytosed magnetic nanoparticles, thereby promoting the development of tissue-engineered tracheal cartilage. After 6 weeks, the tissue-engineered trachea with vascular pedicles can be replaced by end-to-end anastomosis with the autologous trachea.

[0020] In the tissue-engineered trachea of ​​the present invention, the concentration of magnetic nanoparticles and the magnetic induction intensity and frequency of the external magnetic field during use are key factors affecting the cartilage formation process. By changing different magnetic nanoparticle concentrations and external magnetic field parameters, the cartilage formation process of chondrocytes can be affected. In order to make the cartilage rings of the tissue-engineered trachea closer to natural tracheal cartilage in structure and function, the above-mentioned optimal concentration of magnetic nanoparticles and the external magnetic field parameters used in conjunction with them are explored.

[0021] The present invention has the advantages that:

[0022] The present invention combines magnetic nanoparticles with chondrocytes and constructs a magnetic-mechanical force responsive tissue-engineered trachea through 3D printing technology. When co-incubated with cells, magnetic nanomaterials can enter the cells through cell endocytosis, and produce mechanical movement under the action of an external dynamic magnetic field, thereby generating mechanical force on the intracellular structure. Subsequently, when the cells are subjected to external forces at the piconewton level, the mechanical sensitive channels are activated to regulate their related physiological processes. Through remote non-contact mechanical stimulation of chondrocytes, high-strength cartilage is formed as the mechanical skeleton of the tissue-engineered trachea. Under magnetic-mechanical stimulation, the mechanical properties of the cartilage of the tissue-engineered trachea of ​​the present invention are better than those of the simple chondrocyte group without magnetic-mechanical stimulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 . Schematic diagram of the preparation method of magnetic-mechanical force responsive tracheal cartilage rings.

[0024] Figure 2 . Schematic diagram of magnetic-mechanical responsive tissue-engineered trachea.

[0025] Figure 3 .Gross photographs and histological staining photographs of cartilage patches.

[0026] Figure 4 .Cartilage biochemical indicators and mechanical test results.

[0027] Figure 5 .Expression levels of genes related to bone cell extracellular matrix secretion. DETAILED DESCRIPTION

[0028] The specific implementation methods provided by the present invention are described in detail below in conjunction with examples.

[0029] Example 1: Construction of tissue engineered trachea

[0030] The magnetic nanoparticles were directly immersed in 75% ethanol for 30 minutes before use; they were irradiated with ultraviolet light for 30 minutes to fully sterilize them.

[0031] The photoinitiator phenyl-2,4,6-trimethylbenzoyl lithium phosphite (LAP) was dissolved in PBS solution to prepare a 0.25% (w / v) initiator standard solution. Subsequently, methacrylated gelatin (GelMA) was dissolved in the initiator standard solution and heated in a water bath until completely dissolved to form a 10% (w / v) methacrylated gelatin solution. After the temperature of the GelMA solution dropped to room temperature, the chondrocyte suspension was mixed with the GelMA solution at a concentration of 1×108 / mL, and then the magnetic nanoparticles were added to the above mixed system at a concentration of 12.5μg / mL.

[0032] The mixed system prepared in the above steps is printed into tissue-engineered tracheal cartilage rings (the inner and outer diameters of the tissue-engineered tracheal cartilage rings are determined according to the inner and outer diameters of the trachea reconstructed by CT scan of the experimental animal) by a 3D bioprinter under 400-408nm ultraviolet light. Multiple tracheal cartilage rings are stacked layer by layer to form a tissue-engineered trachea with a structure similar to that of a natural gas tube.

[0033] After being embedded in the rabbit body, a magnetic field was applied to the experimental rabbit for treatment (the magnetic induction intensity was 260mT, the frequency was 1Hz, the treatment time was 20min / d, and the magnetic field type was a rotating magnetic field). The chondrocytes that had endocytosed the magnetic nanoparticles responded to the in vitro rotating magnetic field and activated the intracellular mechanical signaling pathway, and the expression of chondrogenesis-related genes in chondrocytes increased, thereby enhancing their chondrogenesis process ( Figure 5 ).

[0034] The present invention combines magnetic nanoparticles with an external rotating magnetic field to construct a magnetic-mechanical force responsive tissue engineering trachea, and forms high-strength cartilage as the mechanical skeleton of the tissue engineering trachea through remote non-contact mechanical stimulation of chondrocytes.

[0035] Example 2: Construction of tracheal patch structure

[0036] The mixed system described in Example 1 was printed into a 1*1.5 cm GelMA patch and transplanted into nude mice for 2 weeks. During this process, magnetic field stimulation was performed as in Example 1 to sample and analyze cartilage growth.

[0037] The effect of the system was preliminarily verified by subcutaneous transplantation of the tracheal patch structure in nude mice. Paraffin sections were prepared 14 days after subcutaneous embedding in nude mice, and HE staining was performed to evaluate the morphology of chondrocytes and cartilage matrix secretion.

[0038] The results showed that chondrocytes were evenly distributed and formed specific cavity-like cartilage pit structures similar to those in the natural state ( Figure 3). The content of glycosaminoglycan in cartilage was quantified using biochemical and molecular biological detection kits. The results showed that the content of glycosaminoglycan in cartilage was higher than that in the simple chondrocyte group without magnetic-mechanical stimulation. The samples were compressed using a low-force universal testing machine, and the Young's modulus was calculated based on the stress-strain curve of the samples. The results showed that under magnetic-mechanical stimulation, the mechanical properties of cartilage were better than those of the simple chondrocyte group without magnetic-mechanical stimulation ( Figure 4 ).

[0039] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A method for preparing a magnetic-mechanical force responsive tissue engineering trachea, characterized in that: The following steps are involved: (A) First, the photoinitiator phenyl-2,4,6-trimethylbenzoyl lithium phosphite was dissolved in PBS solution to prepare a 0.25% (w / v) initiator standard solution. Then, methacryloyl gelatin was dissolved in the initiator standard solution and heated in a water bath until it was completely dissolved to form a 10% (w / v) methacryloyl gelatin solution. After the temperature dropped to room temperature, the chondrocyte suspension was incubated at 1×10 8 / mL concentration was mixed with methacrylated gelatin solution, and then magnetic nanoparticles were added to the mixed system at a concentration of 12.5 μg / mL; (B) The mixed system prepared in the above steps is printed into tissue-engineered tracheal cartilage rings by a 3D bioprinter under 400-408nm ultraviolet light irradiation; multiple tracheal cartilage rings are stacked layer by layer to form a tissue-engineered trachea.

2. The method for preparing the magnetic-mechanical force responsive tissue engineering trachea according to claim 1, characterized in that: The magnetic nanomaterial is Fe3O4 nanoparticles with an average hydrodynamic diameter of about 200nm; the magnetic nanoparticles are directly immersed in 75% ethanol for 30 minutes before use; and are irradiated with ultraviolet light for 30 minutes to be fully sterilized.

3. The method for preparing the magnetic-mechanical force responsive tissue engineering trachea according to claim 1, characterized in that: The method for preparing the chondrocyte suspension comprises the following steps: The cartilage was minced into 1 mm × 1 mm pieces, and 15% type II collagenase was added. The cells were digested at 37°C for 24 h, with several shakes and blows. The cells were filtered through a 0.22 μm sterile filter, centrifuged, washed, resuspended, and counted. The cells were counted at 1.0 × 10 4 / cm 2 Cells were seeded at a density of 10 cm 2 The cells were cultured in a culture dish with a cartilage proliferation medium containing serum and antibiotics; the culture was carried out at 37°C, 5% CO2, and saturated humidity. The medium was changed for the first time on the third day, and then replaced with fresh cartilage proliferation medium every other day; when the cells basically covered the culture dish, about 1.5 mL of trypsin digestion solution was added. After the cell cytoplasm shrank and the morphology became round under the microscope, DMEM culture medium was added to terminate the digestion, the cells were collected, resuspended after centrifugation, and then 1.0×10 4 / cm 2 The cells were seeded at a new 10 cm 2 The culture dish is prepared, and the above steps are repeated. When the cells are expanded to the second generation, the cells are cultured for 3-5 days until the cells fill the culture dish, and the cells are collected to obtain the chondrocyte suspension.

4. The method for preparing the magnetic-mechanical force responsive tissue engineering trachea according to claim 3, characterized in that: The cartilage is rabbit ear cartilage.

5. A magnetic-mechanical force responsive tissue engineering trachea, characterized in that: The method is described in any one of claims 1 to 4.

6. Use of the magnetic-mechanical force responsive tissue engineered trachea as claimed in claim 5 in the preparation of products for reconstruction and regeneration of airway defects.

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