CN114940971B - Construction method of high-activity cartilage tissue based on Faraday wave - Google Patents
Construction method of high-activity cartilage tissue based on Faraday wave Download PDFInfo
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- CN114940971B CN114940971B CN202210726889.6A CN202210726889A CN114940971B CN 114940971 B CN114940971 B CN 114940971B CN 202210726889 A CN202210726889 A CN 202210726889A CN 114940971 B CN114940971 B CN 114940971B
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Abstract
The invention discloses a method for constructing high-activity cartilage tissue based on Faraday waves. The method comprises the steps of inoculating seed cells for constructing cartilage tissues into a microcarrier, constructing a cell-carrying module, and utilizing Faraday wave sound field to drive the cell-carrying module to assemble, fix and culture. By utilizing the method disclosed by the invention, not only can the engineered cartilage tissue be prepared, but also the activity of the obtained cartilage tissue is high. The invention overcomes the defect that the prior Faraday wave assembly method is not suitable for constructing cartilage tissues, and simultaneously provides a novel construction method for engineering sources of high-activity cartilage tissues, and has simple operation and good practicability.
Description
Technical Field
The invention belongs to the technical field of tissue engineering. And more particularly to a method for constructing a high-activity cartilage tissue based on faraday waves.
Background
Cartilage tissue is composed of chondrocytes, matrix and fibers, and has limited self-repair ability because the cartilage tissue does not contain blood vessels, nerves and lymph tissues, and the appearance of engineered cartilage tissue provides a new scheme for repair and treatment of cartilage injury.
The construction strategies of the engineering cartilage tissue mainly comprise a top-down strategy and a bottom-up strategy. The method for constructing the cartilage tissue by inoculating the cells and the growth factors to the macroscopic scaffold adopts a construction strategy from top to bottom, and has the defects of low cell density, difficult material transportation, uneven cell distribution and the like. The method for constructing cartilage tissue by using cell, micro-tissue or cell-carrying module as construction unit and biological printing or biological assembling has no said defect, so that it is an important means for constructing engineering cartilage tissue.
3D bioprinting is the most widely applied assembly method in a bottom-up strategy, can realize ordered arrangement of various cells, and has natural advantages in constructing a gradient cartilage structure; but the shearing force generated in the 3D biological printing process can damage cells, reduce cell activity, and the printing process is long in time consumption, low in efficiency when printing large-size tissues, and limits the application of the printing process. Compared with 3D biological printing, faraday wave biological assembly can assemble microscale materials or cell modules into specific shapes in a non-contact and non-invasive mode, can realize the assembly of in-field modules within a few seconds, has higher efficiency, and is more advantageous in constructing high-activity engineering tissues. For example, chen et al assembled cell spheres composed of hepatocytes, fibroblasts and human umbilical vein endothelial cells using faraday waves, resulting in highly active liver tissue (Chen P, et al adv Healthcare mate, 2015,4,1937-1943). Vahis Serploshan et al utilize Faraday waves to drive myocardial cytoballs to rapidly aggregate to form heart micro-tissues, the assembled cell activity and metabolic level are maintained at high levels, and the assembled heart micro-tissues have the function of natural myocardial tissue contraction and relaxation (Serploshan V, et al biomaterials,2017,131,47-57).
Although the prior art has reported successful assembly of liver tissue and myocardial tissue using Faraday waves, corresponding cytoballs are required to be obtained first. Unlike high cell density tissues such as liver tissue and myocardial tissue, the cartilage tissue is the most abundant in extracellular matrix (ECM) and contains only a small amount of chondrocytes. Therefore, the existing method for obtaining high-density tissue by assembling cytoballs using faraday waves is not suitable for construction of engineered cartilage tissue.
Disclosure of Invention
The invention aims to overcome the defect and the defect that the existing Faraday wave assembly method is not suitable for constructing engineering cartilage tissues, and provides a method for constructing high-activity cartilage tissues based on Faraday waves.
The first object of the present invention is to provide a method for constructing a highly active cartilage tissue based on Faraday waves.
A second object of the present invention is to provide an engineered highly active cartilage tissue constructed by the method.
The third object of the invention is to provide the application of the constructed engineering high-activity cartilage tissue in cartilage injury repair, construction of cartilage disease model or drug screening.
The above object of the present invention is achieved by the following technical scheme:
the invention provides a construction method of high-activity cartilage tissue based on Faraday waves, which comprises the following steps:
s1, inoculating seed cells for constructing cartilage tissues into a microcarrier to construct a cell-carrying module;
s2, uniformly dispersing the cell-carrying modules obtained in the step S1 in an assembly driving liquid, and driving the cell-carrying modules to be assembled, fixed and cultured by utilizing a Faraday wave sound field.
Alternatively, the seed cells of step S1 are bone marrow mesenchymal stem cells, myeloma cells or chondrocytes.
Specifically, the myeloma cell is a mouse myeloma cell ATDC5.
Specifically, the bone marrow mesenchymal stem cells are rat bone marrow mesenchymal stem cells rBMSCs.
In an alternative embodiment, the microcarrier in step S1 is one or more of a gelatin-based porous microsphere, a collagen-based porous microsphere, a hyaluronic acid-based porous microsphere, and a chondroitin sulfate-based porous microsphere.
As a preferred embodiment, the microcarrier used in step S1 is a gelatin-based porous microsphere with a size of 200-300 μm and a pore size of 10-30. Mu.m.
The present invention has found that the cells loaded with the gelatin-based porous microspheres have higher activity in the case of expression of chondrocyte marker genes closer to chondrocytes than the cells not loaded with the gelatin-based porous microspheres. From the assembly results, the engineered cartilage tissue can be assembled by using cells loaded on the gelatin-based porous microspheres through a Faraday wave driven assembly method, and the engineered cartilage tissue has high activity similar to the natural cartilage tissue in composition and structure. Cartilage tissue contains a large amount of extracellular matrix (ECM) in addition to a small amount of chondrocytes, and ECM plays an important role in the structure, mechanical properties and physiological functions of cartilage tissue, which is a non-negligible important feature in engineering cartilage tissue. The aggregated gelatin-based microspheres can serve as a rich ECM, such as single cells, micro-tissue blocks, organoids, cytoballs, etc., as an assembly unit, and even if an engineered tissue can be successfully assembled, the composition and structure of the aggregated gelatin-based microspheres are obviously different from those of natural cartilage tissues and do not have high activity due to the lack of sufficient ECM.
Specifically, 50 to 200 cells are inoculated onto the single microcarrier in step S1.
Specifically, the diameter of the cell-carrying module in step S1 is smaller than 1mm.
Specifically, in step S2, the assembly driving solution is a methacrylic gelatin solution, and after assembly, the assembly driving solution is fixed by irradiation of blue light.
The methacryloylated gelatin (GelMa) is double bond modified gelatin, the methacryloylated gelatin used in the invention is GelMa-30, and the degree of substitution of amino groups after modification is 30+/-5%.
More specifically, the methylpropionated gelatin solution has a mass volume concentration of 8% (w/v); the time for irradiating blue light was 40s.
Specifically, in step S2, the cell-carrying modules and the assembly driving solution are used in an amount of 1.2mL of the assembly driving solution dispersed in every 2.5 ten thousand cell-carrying modules.
Specifically, the driving conditions of the faraday-wave sound field driving in step S2 are as follows: the sine wave has the frequency of 80Hz, and the amplitude is 2-3V after being amplified by the power amplifier.
Specifically, the assembly chamber used in step S2 is cylindrical in shape, 20mm in diameter and 1.5mm in depth.
Specifically, the culturing in the step S2 comprises common culturing and induced culturing of the assembled cell-carrying module; if all the seed cells of the cartilage tissue constructed by the construction method are myeloma cells or bone marrow mesenchymal stem cells, after the assembly and fixation are completed, the cells are cultured in a complete culture medium for 7 days to proliferate, and then the culture medium is replaced by a cartilage differentiation culture medium for cartilage induction culture.
Specifically, the chondrogenic differentiation medium used for myeloma cell ATDC5 was: F12/DEME basal medium, 10% fetal bovine serum, 1% diabody and 1×ITS.
Specifically, the chondrogenic differentiation medium used for bone marrow mesenchymal stem cells rBMSCs is: F12/DEME basal medium, 10% fetal bovine serum, 1% diabody, 1×ITS, 10ng/mL bFGF, 10ng/mL TGF-. Beta.1, 100nM/L dexamethasone (molecular weight: 392.46100), 40g/mL proline, 50g/mL ascorbic acid.
The invention also applies for protecting the engineering high-activity cartilage tissue constructed by the method and the application of the cartilage tissue in cartilage injury repair, cartilage disease model construction or drug screening.
The invention has the following beneficial effects:
the invention provides a construction method of high-activity cartilage tissue based on Faraday waves, by using the method, not only can the engineered cartilage tissue be prepared, but also the activity of the obtained cartilage tissue is high. The invention overcomes the defect that the prior Faraday wave assembly method is not suitable for constructing cartilage tissues, and simultaneously provides a novel construction method for engineering sources of high-activity cartilage tissues, and has simple operation and good practicability.
Drawings
Fig. 1 is a flowchart of a method for constructing a high-activity cartilage tissue based on faraday waves according to the present invention.
FIG. 2 shows the results of staining live and dead cells of mice-carrying myeloma cell microspheres after various days of assembly and fixation culture.
Fig. 3 shows the results of quantitative experiments on cell proliferation in mice-carrying myeloma cell microspheres after various days of assembly and fixation culture, where p < 0.001 is represented.
FIG. 4 shows the results of cytoskeletal and nuclear staining of mouse myeloma cells after 0, 14, 28 days of induced chondrogenic differentiation.
Fig. 5 shows the results of RT-qPCR assays after 0, 7, 14, 21 and 28 days of induction of chondrogenic differentiation in myeloma cells in mice, where p < 0.05 is represented by p < 0.01 and p < 0.001 is represented by p.
FIG. 6 shows the results of staining live and dead cells, cytoskeleton and nuclei of mouse-bearing myeloma cell microspheres after 28 days of assembly and fixation induced chondrogenic differentiation culture; wherein, the upper graph shows the staining results of live and dead cells, and the lower graph shows the staining results of cytoskeleton and nuclei.
FIG. 7 shows the staining results of live and dead cells after mice myeloma cells ATDC5 were inoculated into gelatin-based porous microspheres for different days.
FIG. 8 shows the number and distribution of cells at different depths after the mouse myeloma cells ATDC5 were inoculated into the gelatin-based porous microspheres for 7 days.
FIG. 9 shows the results of two-dimensional plate culture of mesenchymal stem cells and the measurement of Prg4 gene expression amount of mesenchymal stem cells loaded on gelatin-based porous microsphere, wherein p < 0.05 and p < 0.001 are shown.
FIG. 10 shows the result of staining live and dead cells in cell-loaded microspheres after fixation with GelMa solutions of different concentrations.
FIG. 11 shows the results of live and dead cell staining of bone marrow-loaded mesenchymal stem cell microspheres after various days of assembly and fixation culture.
Fig. 12 shows the proliferation of cells in bone marrow-carrying mesenchymal stem cell microspheres after various days of assembly and fixation culture, wherein p < 0.001 is represented.
FIG. 13 shows the results of cytoskeletal and nuclear staining of bone marrow mesenchymal stem cells after 0, 14, 28 days of induced chondrogenic differentiation.
Fig. 14 shows the results of RT-qPCR assays after 0, 7, 14, 21 and 28 days of induction of bone marrow mesenchymal stem cells into cartilage differentiation, where p < 0.05 and p < 0.001.
FIG. 15 shows the results of staining live and dead cells, cytoskeleton and nuclei of bone marrow-loaded mesenchymal stem cell microspheres after 28 days of assembly and fixation induced chondrogenic differentiation culture; wherein, the upper graph shows the staining results of live and dead cells, and the lower graph shows the staining results of cytoskeleton and nuclei.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
Example 1 method for constructing Faraday wave-based high-Activity cartilage tissue
The construction method of the Faraday wave-based high-activity cartilage tissue comprises the following steps:
s1, inoculating seed cells for constructing cartilage tissues into a microcarrier to construct a cell-carrying module;
s2, uniformly dispersing the cell-carrying modules obtained in the step S1 in an assembly driving liquid, and driving the cell-carrying modules to be assembled, fixed and cultured by utilizing a Faraday wave sound field.
In this example, the mouse myeloma cells ATDC5 and gelatin-based porous microspheres are taken as examples, and the construction method of the faraday-wave-based high-activity cartilage tissue of the present invention is described in detail, and the flow of the construction method is shown in fig. 1.
1. Construction of cell-carrying Module
The method comprises the following specific steps:
(1) Inoculating: preparing a mouse myeloma cell ATDC5 cell suspension, uniformly adding the obtained cell suspension to a microcarrier, and completely immersing the cell suspension into the microcarrier; the microcarrier used in this example was a gelatin-based microcarrier of the shrine organism (3D TableTrix TM Microchip sheet TM The product number is F01-100), which can be dispersed into tens of thousands of single gelatin-based porous microspheres after absorbing the culture medium in the cell suspension, and no redundant cell suspension flows out after the cell suspension is absorbed; this example inoculates 100 cells on a single microcarrier (gelatin-based porous microsphere);
(2) And (3) wall-attached culture: after cell inoculation is finished, 2mL of PBS is dripped into the inter-pore gap of the non-TC 6 pore plate, so that the culture solution is prevented from evaporating too fast, and the culture solution is placed into a cell incubator for incubation for 2 hours, so that cells are attached to the wall;
(3) Supplementing liquid: after the cells are attached, adding 8mL of complete culture medium into the holes, and placing the cells into a cell culture box for culture; when the culture medium is added, the microcarrier which is inoculated with cells is prevented from being directly washed by the culture medium, after the culture medium is added, the microcarrier can be gently disturbed by a gun head to ensure that the microcarrier is uniformly distributed, the microcarrier is not required to be completely scattered, the diameter of a microcarrier block (a cell carrying module) is not more than 1mm, and the diameter of the cell carrying module is 200-500 mu m;
(4) Liquid replacement: and standing the culture plate for 2-5 minutes, slightly tilting the culture plate after all the microcarriers are sunk into the bottom of the hole, discarding 50-70% of culture medium, supplementing 8mL of fresh culture medium, and assembling after ensuring that cells are adhered to the porous microspheres.
When the culture medium is replaced according to the culture requirement, after all microcarriers settle at the bottom of the hole, carefully discarding the culture medium, avoiding microcarrier loss, and adding fresh culture medium.
2. Faraday wave driven assembly
The invention finally selects the methacryloylated gelatin (GelMa) as driving liquid and fixing liquid in the Faraday wave driving assembly process through pre-experimental screening. The methacryloylated gelatin (GelMa) is double bond modified gelatin, the methacryloylated gelatin used in the invention is GelMa-30, and the degree of substitution of amino groups after modification is 30+/-5%.
The preparation method of the GelMa-30 solution comprises the following steps:
(1) Preparing 0.25% (w/v) initiator standard solution
10mL of PBS solution was added to a brown bottle (containing 0.025g LAP) containing initiator (LAP); heating and dissolving in water bath at 40-50 deg.c for 15 min and oscillating several times;
(2) Preparing GelMa solution
Placing GelMa with required mass into a container (centrifuge tube/glass bottle/beaker), dissolving with initiator standard solution at room temperature, sterilizing the GelMa solution with 0.22 μm sterile needle filter, and preserving in dark for subsequent cell culture and assembly experiment.
The specific process of Faraday wave driven assembly is as follows:
taking the cell-carrying module obtained after culturing for 24 hours in the embodiment 1 (at the moment, cells are adhered to the porous microspheres) as an assembling unit, uniformly dispersing the cell-carrying module in GelMa solution, adding 2.5 ten thousand cell-carrying modules into 1.2mL of assembling driving liquid, transferring the GelMa solution containing the cell-carrying modules into an assembling chamber (the shape of the assembling chamber used in the embodiment is cylindrical, the diameter is 2.0mm and the depth is 1.5 mm) of a Faraday wave driving device, standing, setting driving conditions (the driving condition of Faraday waves is sine waves, the frequency is 80Hz and the amplitude is 2-3V after being amplified by a power amplifier), driving the cell-carrying modules to be assembled into a pattern by utilizing Faraday wave sound field, and fixing the pattern to form gel by blue light irradiation (40 s), thereby fixing the assembling result; the fixed cell-carrying modules are transferred to a complete culture medium for 7 days to be cultured, and after the cells proliferate for a period of time, the ATDC5 cells are induced to differentiate into cartilage and the differentiation level is characterized.
The induced chondrogenic differentiation process is as follows:
after the assembled cell-carrying microspheres are fixed, culturing the microspheres in a complete culture medium for 7 days, changing the culture medium into a cartilage differentiation culture medium once every 2 days when the number of cells is large, inducing the cartilage differentiation of the microspheres in vitro, and detecting the change of cell phenotype and gene expression quantity through cytoskeleton and cell nucleus staining and RT-qPCR.
The chondrogenic differentiation medium used for the mouse myeloma cells ATDC5 comprises the following components: F12/DEME basal medium, 10% fetal bovine serum, 1% diabody and 1×ITS.
3. Experimental results
Prior to induction of chondrogenic differentiation, the invention detects cell activity and proliferation in assembled and fixed cell-loaded microspheres cultured with complete medium for different days (1, 3, 5, 7 d) by live dead cell staining test (cell live dead staining kit manufacturer: EFL, cat#: EFL-CLD-001) and CCK8 kit (biosharp, cat#: BS 350B-500T). The staining results of live and dead cells of the mice-carrying myeloma cell microspheres after assembly and fixation culture for different days are shown in fig. 2, wherein the live cells are stained green by calcein, and the dead cells are stained red by propidium iodide. As can be seen from the results shown in FIG. 2, the number of dead cells gradually decreased and the number of living cells gradually increased with the increase of the culture time. The quantitative test results of cell proliferation (CCK 8 kit) in the mice-carrying myeloma cell microspheres after assembling and fixing culture for different days are shown in FIG. 3, and the results shown in FIG. 3 show that the assembled and fixed cells maintain a high proliferation rate. The results show that the cells assembled by the method have better activity and proliferation rate.
According to the invention, cell skeletons and cell nucleus staining experiments are carried out on cells subjected to cartilage-forming induced differentiation culture for different days by using rhodamine-labeled Phalloidin (TRITC Phaliodin) and 4, 6-diamidine-2-phenylindole (DAPI), so that the phenotypic change of the cells is observed. Wherein rhodamine labeled Phalloidin (TRITC Phaliodin) stains actin (cytoskeleton) red and 4, 6-diamidine-2-phenylindole (DAPI) stains nuclei blue. The results of cytoskeletal and nuclear staining of mouse myeloma cells ATDC5 after 0, 14, and 28 days of induced chondrogenic differentiation are shown in fig. 4, and it is apparent from the results shown in fig. 4 that ATDC5 cells gradually round from the original spindle shape and approach the cell morphology of chondrocytes as the differentiation time is prolonged.
The Col2a1 gene codes for the alpha-1 chain of type II collagen which exists in cartilage and vitreous, and the gene mutation is related to diseases such as dysplasia of cartilage, early-onset familial osteoarthritis and the like; the Aggrecan (abbreviated as Agg, also called ACAN) gene codes for cartilage specific proteoglycan core protein which is the main component of extracellular matrix of cartilage tissue and is mainly used for resisting compression of cartilage; the Prg4 gene encodes proteoglycan 4, a highly glycosylated secreted mucin-like protein, highly expressed in synovial tissue, cartilage and liver, exerts boundary lubrication in joints, and prevents deposition of proteins from synovial fluid onto cartilage by controlling adhesion-dependent synovial growth and inhibiting adhesion of synovial cells to cartilage surfaces; the Sox9 gene encoded protein plays a role in the chondrocyte differentiation process, and the lack can lead to skeletal deformity syndrome; the increase of the expression level of the four genes can prove that the bone marrow mesenchymal stem cells and the myeloma cells differentiate towards the direction of the chondrocytes. Therefore, the present invention characterizes cells after chondrogenic induction differentiation by detecting changes in the expression levels of the four genes described above.
The results of RT-qPCR test of mouse myeloma cells ATDC5 after induction of chondrogenic differentiation for 0, 7, 14, 21 and 28 days are shown in FIG. 5, the relative expression level of the target gene of undifferentiated ATDC5 is set to 1, and the results of RT-qPCR test shown in FIG. 5 show that the expression levels of type II collagen (Col 2a 1) and proteoglycan (Aggrecan) are remarkably increased along with the prolongation of differentiation time, the genotypes of the cells are similar to that of chondrocytes, and the chondrogenic differentiation degree of the cells is gradually increased along with the prolongation of culture time after the induction of chondrogenic differentiation medium is added.
The results of live cell staining and cytoskeleton and nucleus staining of the mice-carrying myeloma cell microsphere after 28 days of assembly and fixation induced chondrogenic differentiation are shown in fig. 6, and the results shown in fig. 6 show that the porous microsphere after 28 days of culture is tightly connected together through cells and maintains higher cell activity, which indicates that the method can be used for obtaining high-activity tissues.
The results show that the method can be used for constructing myeloma cells into high-activity engineering cartilage tissues.
Example 2
According to the invention, the mouse myeloma cells ATDC5 are inoculated to the gelatin-based porous microspheres by the method, and then are respectively cultured for 1, 3, 5 and 7 days, and the activity and proliferation condition of cells on the microspheres are observed through a living and dead cell staining experiment. After 7 days of incubation, the number and distribution of cells at different depths of the microspheres were also observed by a confocal laser microscope, i.e. from the top of the porous microspheres, the number and distribution of cells at 10, 20, 30, 40, 50 and 60 μm cross-sections were observed, respectively.
The staining results of the living cells of the mice myeloma cells ATDC5 inoculated on the gelatin-based porous microspheres for different days are shown in FIG. 7, and the results of FIG. 7 show that the number of the living cells is large, the number of the dead cells is small, and the number of the cells is obviously increased along with the extension of the culture days, so that the gelatin-based porous microspheres have no cytotoxicity and can maintain the proliferation of the cells. The number and distribution of cells at different depths of the gelatin-based porous microspheres are shown in FIG. 8, and it is apparent from the results shown in FIG. 8 that cells exist not only on the surface of the microspheres but also in the interior of the microspheres, indicating that cells can migrate into the interior of the microspheres. The above results indicate that the gelatin-based porous microspheres are favorable for cell adhesion, growth, proliferation and migration, thereby obtaining cell-carrying microspheres carrying a sufficient number of cells, and being used as assembly modules for subsequent experiments.
The invention detects the expression level of cell Prg4 gene (cartilage cell marker gene) after the bone marrow mesenchymal stem cells inoculated in gelatin-based porous microspheres and the bone marrow mesenchymal stem cells cultured by two-dimensional flat plates are placed under the same culture condition and respectively induced to differentiate and culture for 0, 7, 14, 21 and 28 days. The results of measuring the expression level of Prg4 gene in bone marrow mesenchymal stem cells cultured in two-dimensional plates and bone marrow mesenchymal stem cells loaded in gelatin-based porous microspheres are shown in FIG. 9, and it is clear from the results shown in FIG. 9 that the expression level of chondrocyte marker gene in bone marrow mesenchymal stem cells loaded in gelatin-based porous microspheres is closer to that in chondrocytes and the cells exhibit higher activity than in cells grown in two-dimensional plates. From the assembly results, the engineered cartilage tissue can be assembled by using cells loaded on the gelatin-based porous microspheres through a Faraday wave driven assembly method, and the engineered cartilage tissue has high activity similar to the natural cartilage tissue in composition and structure. The cartilage tissue contains a large amount of extracellular matrix (ECM) besides a small amount of cartilage cells, and the ECM plays an important role in the structure, mechanical property and physiological function of the cartilage tissue and is a non-negligible important characteristic in engineering construction of the cartilage tissue. The aggregated gelatin-based microspheres can serve as a rich ECM, such as single cells, micro-tissue blocks, organoids, cytoballs, etc., as an assembly unit, and even if an engineered tissue can be successfully assembled, the composition and structure of the aggregated gelatin-based microspheres are obviously different from those of natural cartilage tissues and do not have high activity due to the lack of sufficient ECM.
Example 3
This example examined the effect of the concentration of fixative (methacryloylated gelatin solution) on cell activity.
According to the preparation method of the GelMa-30 solution disclosed by the embodiment 1, gelMa solutions with the mass and volume percentage (w/v) concentration of 6%, 7%, 8%, 9% and 10% are respectively prepared, after patterns are formed by driving assembly by utilizing Faraday wave sound fields, the GelMa solutions with different concentrations are respectively used for fixation, the culture is carried out for 24 hours, and the cell activity is observed through a live dead cell staining test. As shown in FIG. 10, the result of staining live and dead cells in the cell-carrying microspheres after fixation with GelMa solutions of different concentrations shows that when the concentration is 8% (w/v), only a small amount of dead cells appear and the cell activity is good, so that the concentration of the fixation solution is 8% (w/v), and the same concentration is used for assembling the driving solution.
Example 4
In this example, rat bone marrow mesenchymal stem cells rBMSCs were used as seed cells, and engineered cartilage tissue was constructed by the construction method described in example 1, except that the cells used and the culture medium for inducing cartilage differentiation were different, and the conditions were the same as in example 1, except that the concentration of driving solution and fixing solution, i.e., gelMa, was 8% (w/v).
The cell activity and proliferation of bone marrow mesenchymal stem cells in assembled and fixed cell-carrying microspheres cultured with complete medium for different days (1, 3, 5, 7 d) were detected by a live dead cell staining test and CCK8 kit in this example before inducing chondrogenic differentiation. The results of staining live cells of bone marrow-loaded mesenchymal stem cell microspheres assembled and cultured for different days are shown in fig. 11, and as shown in fig. 11, the number of dead cells gradually decreases and the number of live cells gradually increases with the extension of the culture time, indicating that the cell activity is good. As shown in fig. 12, the proliferation test results of the cells in the bone marrow-loaded mesenchymal stem cell microspheres after assembling and fixing culture for different days show that the assembled and fixed cells maintain a high proliferation rate as shown in fig. 12. The results show that the bone marrow mesenchymal stem cells assembled by the method have better activity and proliferation rate.
The results of cytoskeletal and nuclear staining of bone marrow mesenchymal stem cells after 0, 14 and 28 days of induced chondrogenic differentiation are shown in fig. 13, and it is apparent from the results shown in fig. 13 that the bone marrow mesenchymal stem cells gradually round from the original spindle shape with the extension of differentiation time, and approach to the cell morphology of chondrocytes, indicating that the phenotype of bone marrow mesenchymal stem cells approaches to chondrocytes with the extension of induced differentiation time.
The results of RT-qPCR measurements after 0, 7, 14, 21 and 28 days of induction of chondrogenic differentiation of bone marrow mesenchymal stem cells are shown in FIG. 14, and it is apparent from the results of RT-qPCR measurements shown in FIG. 14 that the expression levels of Col2a1 and Prg4 significantly increase with the prolongation of differentiation time, and the genotypes of the cells tended to chondrocytes, indicating that the degree of chondrogenic differentiation of the cells gradually increased with the prolongation of the induction of differentiation culture time.
The results of live dead cell staining and cytoskeletal and cell nucleus staining of bone marrow-carrying mesenchymal stem cell microspheres after 28 days of assembly and fixation induced chondrogenic differentiation are shown in fig. 15, wherein the upper graph shows the results of live dead cell staining and the lower graph shows the results of cytoskeletal and cell nucleus staining. As shown in FIG. 15, the porous microspheres after 28 days of culture are tightly connected together by the bone marrow mesenchymal stem cells and maintain higher cell activity, indicating that the tissue with high activity can be obtained by the method of the present invention.
The results show that the method can be used for constructing the mesenchymal stem cells to obtain the high-activity engineering cartilage tissue.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (4)
1. The method for constructing the high-activity cartilage tissue based on Faraday waves is characterized by comprising the following steps of:
s1, inoculating seed cells for constructing cartilage tissues into a microcarrier to construct a cell-carrying module; the seed cells are bone marrow mesenchymal stem cells, myeloma cells or chondrocytes; the microcarrier is a gelatin-based porous microsphere;
s2, uniformly dispersing the cell carrying modules obtained in the step S1 in an assembly driving liquid, and driving the cell carrying modules to be assembled, fixed and cultured by utilizing a Faraday wave sound field; wherein the assembly driving liquid is a methacrylic gelatin solution, and is fixed by irradiating blue light after assembly; the driving conditions of the Faraday wave sound field driving are as follows: the sine wave has the frequency of 80 and Hz, and the amplitude is 2-3V after being amplified by the power amplifier.
2. The method of claim 1, wherein 50 to 200 cells are inoculated onto the microcarriers in step S1.
3. The method of claim 1, wherein the cell-carrying modules in step S1 have a diameter of less than 1mm.
4. The method according to claim 1, wherein the cell-carrying modules and the assembly driving solution in step S2 are used in an amount of 1.2mL per 2.5 ten thousand cell-carrying modules dispersed in the assembly driving solution.
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